Shear-Thinning Therapeutic Composition, and Related Methods

ABSTRACT

A shear-thinning therapeutic composition is provided along with methods of making and using the therapeutic composition.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/333,627 filed Mar. 15, 2019, which is the United States NationalPhase of International Application No. PCT/US2017/051791 filed Sep. 15,2017, and claims the benefit of U.S. Provisional Patent Application No.62/394,991 filed Sep. 15, 2016, each of which is incorporated herein byreference in its entirety.

Provided herein are therapeutic compositions and related methods,specifically a shear-thinning dosage form and related methods.

Proteins are useful therapeutic agents for a variety of purposes. In thecontext of regenerative medicine, proteins and polypeptides play keyroles in tissue regeneration by promoting cell proliferation, migrationand differentiation to restore a diseased or damaged tissue. But theshort half-life of exogenous proteins, particularly growth factors,limits the therapeutic efficacy if administered as free proteins.Heparin binds more than 400 proteins and peptides, and the resultantcomplex can stabilize proteins from proteolysis and prolong theirbioactivity. However, typical heparin-protein complexes are soluble inwater, making spatial and temporal control of delivery difficult.

LAPONITE® has been widely used in food, cosmetic and pharmaceuticalindustries as additives, rheological modifier or active ingredients.LAPONITE® is able to generate a stable nanoplatelet dispersion withuniform particle size (25 nm diameter and 0.92 nm thickness), largesurface area (>350 m² g⁻¹), specific surface charges and certainbioactive properties (Gaharwar A K, et al., Adv Mater. 2013; 25:3329-36and Dawson J I, et al. Clay gels for the delivery of regenerativemicroenvironments. Adv Mater. 2011; 23:3304-8). For example, in severalrecent reports, in vitro studies showed that the LAPONITE® dispersion orgel could facilitate chondrogenic differentiation of human bone marrowstromal cells (hBSMCs) or promote osteogenetic differentiation of humanmesenchymal stem cells (hMSCs) (Gaharwar A K, et al., Adv Mater. 2013;25:3329-36 and Dawson J I, et al. Adv Mater. 2011; 23:3304-8). TheLAPONITE® dispersion can self-organize into “house-of-cards” gel throughface-to-edge interaction with gelation rate dependent on itsconcentration (See FIG. 1).

Such LAPONITE® dispersion can either directly absorb biomolecules fordelivery or complex with other synthetic or natural biopolymers to forminjectable nanocomposite hydrogels for cell or drug delivery. However,when it is used for controlled release of bioactive proteins, the strongabsorption on LAPONITE® particles makes release of the proteinsdifficult, defeating the purpose of controlled release. Though manybiomaterials, such as alginate, collagen, hyaluronic acid and chitosan,have been used to mix with LAPONITE® to form injectable hydrogels fordrug delivery (Xavier J R, et al. Bioactive Nanoengineered Hydrogels forBone Tissue Engineering: A Growth-Factor-Free Approach. ACSNANO. 2015;9:3109-18, Divya Bhatnagar D X, et al. Hyaluronic Acid and Gelatin ClayComposite. Journal of Chemical and Biological Interfaces. 2014; 2:1-11;Li Y, et al. pH sensitive LAPONITE®/alginate hybrid hydrogels: swellingbehaviour and release mechanism. Soft Matter. 2011; 7:6231; and Yang H,et al. Composite Hydrogel Beads Based on Chitosan and LAPONITE®:Preparation, Swelling, and Drug Release Behaviour. Iranian PolymerJournal. 2011; 20:479-90), those biomaterials cannot protect theproteins from proteolysis and prolong their bioactivity.

SUMMARY

A composition and related methods are provided herein to take advantageof the unique properties of heparin and other compositions, such assulfated or sulfamated compositions that bind to therapeutic activeagents (therapeutic agents). According to aspects, a hydrogel isprovided that is assembled from silicate platelet nanoparticle (e.g.,LAPONITE® or other clays), a gelling agent, such as heparin, and atherapeutic agent, such as a heparin-binding angiogenic agent or growthfactor, e.g., fibroblast growth factor-2 (FGF2). The gel israpidly-assembled, for example within one minute—resulting in a shearthinning hydrogel comprising a three-dimensional nanocomposite networkformed in aqueous solution based on non-covalent reversible crosslinks.

In aspects, the shear thinning hydrogel is designed to bear functionalmoieties for forming physical, non-covalent crosslinks, such as hydrogenbonding, ionic interaction, host-guest chemistry, hydrophobicinteraction or combination of multiple physical interactions in onesystem. Gelling agents such as biopolymers, for example and withoutlimitation sulfated or sulfamated polysaccharides or sulfated orsulfamated glycosaminoglycans, can directly bind to silicananoparticles, such as LAPONITE® platelets, to generate injectablehydrogels. The interaction between the gelling agent and the clayparticles form injectable hydrogels having a large surface area,specific surface charges and strong binding abilities to a variety oftherapeutic agents, such as proteins, peptides, and oligopeptides. Incontrast to covalently-crosslinked injectable hydrogels, shear thinninghydrogels are also injectable but do not need any triggers to initiate achemical reaction for in situ gelation—which can exhibit toxicity to thesurrounding tissues by virtue of the cross-linkers and/or thecrosslinking chemistries. In the described compositions, the drugs,e.g., biologics, peptides, proteins, oligopeptides, peptide nucleicacids, or nucleic acids, can be fully loaded into a shear thinning geland the gel-sol transition during injection occurs only at the interfacebetween the hydrogel and needle wall. Thus the drug is less likely toleak out, leading to more precise control over dosage and releasekinetics of the drug.

According to one aspect, a shear-thinning therapeutic composition isprovided. In one aspect, the shear-thinning therapeutic compositioncomprises: silicate platelets; a gelling agent non-covalently linkingthe platelets to form a shear-thinning composition; and a therapeuticagent that binds non-covalently to the gelling agent and that iscomplexed non-covalently with the gelling agent.

In other aspects, the composition comprises natural or syntheticsilicate platelets (e.g., LAPONITE®); a sulfated or sulfamated polymernon-covalently linking the platelets to form a shear-thinningcomposition; and a therapeutic agent that is binding partner of thesulfated or sulfamated polymers or oligomers complexed non-covalentlywith the sulfated or sulfamated polymers or oligomers. Optionally, thecomposition comprises natural or synthetic silicate platelets; a gellingagent that is one or more of a cationic amino acid; an anionic aminoacid; a hydrophilic amino acid; and a polypeptide, optionally anoligopeptide, comprising from 2 to 10 amino acids or from 2 to 6 aminoacids and including a plurality of cationic, anionic, or hydrophilicamino acids non-covalently linking the platelets to form ashear-thinning composition; and a therapeutic agent dispersed within thecomposition and/or complexed non-covalently with the silicate plateletsand/or the one or more of a cationic amino acid; an anionic amino acid;a hydrophilic amino acid; a polypeptide, optionally an oligopeptide,comprising from 2 to 10 amino acids or from 2 to 6 amino acids andincluding a plurality of cationic, anionic, or hydrophilic amino acids.Methods of making the therapeutic composition, and methods of using thetherapeutic composition, for example and without limitation to induceangiogenesis or as a soft tissue filler in a patient, also are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. (A) Schematic illustration of forming “house-of-cards” gel fromLAPONITE® nanoplatelets. (B) Oscillatory time sweep and (C) angularfrequency sweep measurements of LAPONITE® gels at differentconcentrations. (Diamond) 26.6 mg/mL, (Square) 30 mg/mL, (Circle) 35mg/mL and (Triangle) 39.1 mg/mL. All LAPONITE® dispersions were preparedby dispersing LAPONITE® powders in deionized water with vigorouslymagnetic stirring for 2 h and then immediately transferred to a parallelplate for oscillatory time sweep (frequency at 1 rad/s and strain at 1%)and angular frequency sweep tests (strain at 1%) (n=3). The resultsindicate that gelation rate is proportional to LAPONITE® concentration.When LAPONITE® concentration is above 30 mg/mL, it can quickly formgels. The storage modulus (G′) is much greater than loss modulus (G″)throughout the frequency sweep measurements at concentration above 30mg/mL, indicating formation of a stable network through “face-to-edge”interaction. (Cummins H Z., Journal of Non-Crystalline Solids. 2007;353:3891-905).

FIG. 2. Chemical structure of heparin sodium salt. The number averagemolecular weight (Mn) of the heparin used is ca. 20 kDa. Each repeatunit bears four negative charges and approximately 117 negative chargescarry on each chain.

FIG. 3. (A) Schematic illustration of gelation between heparin-proteincomplex and LAPONITE® to form Heparin-LAPONITE® gel within one minutefor sustained release. (B) Oscillatory time sweep and (C) angularfrequency sweep measurements of Heparin-LAPONITE® gels. The gels arecomprised of LAPONITE® at 19 mg/mL with various heparin concentrations(H/L ratio). (Diamond) 1.9 mg/mL (1:10), (Square) 3.8 mg/mL (2:10),(Circle) 7.6 mg/mL (4:10), (Triangle) 15.2 mg/mL (8:10). (D) Modulichange as a function of heparin concentrations. Heparin quickly gelswith LAPONITE® dispersion to form Heparin-LAPONITE® gel upon mixing themat appropriate ratio. At a fixed LAPONITE® concentration, the gelationrate is proportional to heparin concentration. The G′ over G″ throughoutfrequency sweep indicates a stable network structure based on rapidreversible crosslinks, which are not disrupted under suitable shearstress. (E) Viscosity change of Heparin-LAPONITE® gels with differentsolid concentrations as a function of shear rate. All the gels showshear thinning properties, indicating injectability.

FIG. 4. (A) Oscillatory time sweep measurements of Heparin-LAPONITE®gels at different solid concentrations (heparin+LAPONITE®) with H/Lratio fixed at 4:10. (Diamond) 5.8+14.4 mg/mL, (Square) 7.6+19 mg/mL,(Circle) 9.4+23.5 mg/mL and (Triangle) 11.2+28 mg/mL. (B) Theviscoelastic properties of Heparin-LAPONITE® gels at different solidconcentrations as a function of angular frequency. (Diamond) 7.6+19mg/mL, (Square) 9.4+23.5 mg/mL, and (Triangle) 11.2+28 mg/mL. (C) Thefirst data points of G′ and G″ from time sweep measurements are plottedversus the solid concentrations to evaluate the gelation rate. Themoduli are increased steadily as the solid concentration increases withG′ approximately 6-19 times higher than that of G″, indicating morecrosslinks formed at higher solid concentrations. But it reaches maximalG′ and G″ at 36.1 mg/mL (10.3+25.8 mg/mL), implying that too high solidconcentration likely hinders heparin diffusion into the LAPONITE®dispersion and reduce gelation rate.

FIG. 5. (A) Angular frequency sweep measurements of LAPONITE® dispersionat 20 mg/mL with different settling time. (Diamond) Fresh dispersion,(Square) 7 days and (Circle) 20 days. The as-made LAPONITE® dispersionat 20 mg/mL only forms a weak gel after mature gelation, which is ableto convert into low-viscosity dispersion again after being manuallyshaken for ca. 2 min. (B) Oscillatory time sweep measurements of thesettled LAPONITE® dispersion gelled with heparin solution to formheparin-LAPONITE® gels. Settling time: (Diamond) Fresh dispersion,(Square) 20 days, (Triangle) 40 days and (Circle) 180 days. TheLAPONITE® dispersion was settled for up to 180 days, which was manuallyshaken to convert into low-viscosity dispersion again and then mixedwith the heparin solution to form heparin-LAPONITE® gels in a nearlysimilar manner like the fresh dispersion. The H/L ratio ofheparin-LAPONITE® gels was fixed at 4:10 (7.6+19 mg/mL). The resultsshow that the settled LAPONITE® dispersion does not sacrifice anygelation ability with heparin. Frequency sweep measurements show aslightly higher G′ and G″ than that gelled with the fresh dispersion,indicating that the LAPONITE® dispersion can be stably stocked for atleast 6 months and used to gel with heparin. The higher moduli from thesettled LAPONITE® dispersion gelled with heparin is likely attributed tothe self-organization of the settled LAPONITE® particles, which iseasier to be connected by heparin chains when mixing the two components.

FIG. 6. Comparison of the viscoelastic properties of heparin-LAPONITE®gels after incubating with biological solutions for a different timeperiod. (A) 0.9% NaCl biological saline, and (B) cell medium (EBM-2).(Diamond) No incubation, (Square) 10 min, (Circle) 40 min and (Triangle)18 h. The freshly made heparin-LAPONITE® gel with H/L ratio at 4:10(7.6+19 mg/mL) was incubated in biological solutions. Theviscoelasticity of all the gels were then examined by angular frequencysweep measurements. Compare with the fresh heparin-LAPONITE® gel,incubation with biological saline or cell medium shows little impacts onthe viscoelastic properties under different incubation time, indicatinga stable network structure resistant to the interference of ions andother biological components. (C) Time sweep measurements of heparingelled with LAPONITE® dispersions prepared at different pH levels. TheLAPONITE® dispersions were prepared by dispersing in deionized waterwith pH ranged from 2.8 to 10.6 adjusted by 0.1 M HCl and 0.1 M NaOHsolutions. The dispersions were then separately mixed with heparinsolutions at H/L ratio at 4:10 by manually swirling for 1 min and thenimmediately transferred for rheometry test. Time sweep measurements showsimilar gelation kinetics and viscoelasticity at different pH levels,indicating little effects on gelation caused by the tested pHconditions.

FIG. 7. Oscillatory time sweep measurements of heparin-LAPONITE® gelswith H/L ratio fixed at 4:10 (7.6+19 mg/mL) but containing differentcontent of NaCl. (Diamond) 0 wt. %, (Square) 0.1 wt. %, (Circle) 0.3 wt.%, (Triangle) 0.6 wt. % and (Asterisk) 0.9 wt. %. Compare withheparin-LAPONITE® gel without NaCl, the gels containing NaCl ranged from0.1 to 0.9 wt. % show an increased gelation rate with G′ and G″ almosttripled as the NaCl content increased from 0 to 0.9 wt. %. This resultindicates that heparin together with reasonable amount of salt ionsenhanced gelation process, instead of inhibiting or retarding gelation.This is mainly because salt ions promoted LAPONITE® gelation.

FIG. 8. Zeta potential measurements of LAPONITE®, heparin andheparin-LAPONITE® complexes as a function of heparin concentration.(Filled Diamond) LAPONITE® control at 9.5 mg/mL, (Square) heparinsolutions from 0.95 to 7.6 mg/mL, and (Triangle) heparin-LAPONITE®complexes are comprised of heparin and LAPONITE® with concentrationsranged from 0.95+9.5 to 7.6+9.5 mg/mL. The H/L ratio of theheparin-LAPONITE® complexes is same to the heparin-LAPONITE® gels rangedfrom 1:10 to 8:10. The heparin-LAPONITE® complexes show a saturatingabsorption at heparin concentration between 4.75 to 5.7 mg/mL (H/L, 5:10to 6:10). Above this threshold, more free heparin molecules contributeto the zeta potential values with a similar trend as the heparin controlshows.

FIG. 9. (A) Cumulative release of FGF2 from heparin-LAPONITE® gels:(Diamond) 3.8+19 mg/mL, (Square) 7.6+19 mg/mL and (Asterisk) 15.2+19mg/mL; LAPONITE® controls: (Circle) 22.8 mg/mL and (Triangle) 26.6mg/mL. The FGF2 is sustainably released from heparin-LAPONITE® gels over34 days with release rate related to the H/L ratio, but a negligibleFGF2 is released from both LAPONITE® controls. (B, C) Western blotassays to examine the stability of the released FGF2 fromheparin-LAPONITE®, LAPONITE® and hyaluronic acid-LAPONITE®(HA-LAPONITE®) gels with regard to proteolytic degradation. The releasedFGF2 solution is mixed with trypsin (mass ratio, 1:200) and incubated at37° C. for 0.5 and 2 h. The FGF2 from heparin-LAPONITE® gel shows asimilar band intensity compared to heparin-FGF2 complex control,indicating a heparin-binding FGF2 released from heparin-LAPONITE® geland protection from protease degradation. The free FGF2 is readilydegraded by trypsin treatment within 0.5 h. Neither LAPONITE® norHA-LAPONITE® gels yield detectable and stable FGF2 before and aftertreatment with trypsin.

FIG. 10. (A) Micrographs of the H&E stained sections surrounding theinjected heparin-LAPONITE® gel. Low magnification of images of the H&Estained tissues harvested at days (a) 3, (b) 14 and (c) 28, (Scale bar,500 μm). Rectangular frames indicate the region chosen for highermagnifications (d-f), (Scale bar, 100 μm). (B) MTS stained tissuesillustrate collagen deposition (arrow) after heparin-LAPONITE® injectionat days (a) 14 and (b) 28, (Scale bar, 100 μm).

FIG. 11. Representative micrographs of immunohistochemically stainedsections of CD68 positive macrophages merged with DAPI staining (Scalebar, 100 μm). Tissues were harvested at days (A) 3, (B) 14 and (C) 28.(D) The number of CD68 positive macrophages (MP number per mm²) atdifferent time points. The population of CD68+ cells decreasessignificantly from day 3 to week 2, and may have slightly increased from2 to 4 weeks, however, the difference is not statistically significant.Images from more than 5 random areas around the injection site are usedfor quantification. ** P=0.0067, P<0.05 is considered significant. Datarepresent mean±SD (n 5).

FIG. 12. Potent angiogenesis induced by sustained release of FGF2 fromheparin-LAPONITE® gel. (A-C) Representative confocal micrographs showthe distribution of blood vessels in tissues marked by CD31 and SMAstaining (originals in color) (Scale bar, 100 μm). The tissues wereharvested at 2 weeks after injection with (A) heparin-LAPONITE® alone,(B) the contralateral tissue without gel implantation as a control, and(C) FGF2-loaded heparin-LAPONITE® gel. Compare with heparin-LAPONITE®gel alone and its contralateral, FGF2-loaded heparin-LAPONITE® gelefficiently induces generation of more mature blood vessels by thesustainably released FGF2. (D) Comparison of blood vessel number perunit area in the tissues adjacent to the implantation site. The bloodvessel number per square millimeter is calculated by counting the vesselnumber over the area. The free FGF2 control is adopted from a previousstudy conducted by the same person (Chu H, et al., Injectable fibroblastgrowth factor-2 coacervate for persistent angiogenesis. Proc. Nat'l.Acad. Sci. U.S.A. 2011; 108:13444-9). Images from 10-20 random areasaround the injection site are used for quantification. One-way ANOVAfollowed by Bonferroni correction, ***P<0.0001, P<0.05 is consideredsignificant. Data represent mean±SD.

FIG. 13 (original in color). Representative microscopic images of themerged CD31, SMA and DAPI stained sections show cell activities ofendothelial and smooth muscle cells induced by the heparin-LAPONITE® gelimplantation. (A) 2 weeks and (C) 4 weeks post-injection (Scale bar: 500μm). (B and D) are the corresponding higher resolution images of theselected rectangular area (Scale bar: 100 μm). Compare to that of 2weeks post-injection, a significant amount of endothelial cells andsmooth muscle cells are recruited toward the implanted gel at 4 weeksafter injection. These cells trend to migrate from gel edge to center.The two cells accumulated on some areas of the gel edge showangiogenesis. The gel shows less dense at 4 weeks post-injection,indicating the gel degradation by the recruited cells.

FIG. 14. In vivo biodegradation of heparin-LAPONITE® gel with or withoutFGF2 by subcutaneous implantation for 8 weeks. (A) heparin-LAPONITE® gelalone is gradually degraded in 8 weeks. (B) FGF2-loadedheparin-LAPONITE® gel is degraded in about 6 weeks. 100 μL ofheparin-LAPONITE® gel with or without FGF2 (500 ng) was injected in theback of BALB/cJ mice. Without growth factor, the gel is graduallydegraded in 8 weeks without causing mouse death, any malignantinfection, or abscess at the injection sites. When loading with FGF2,the material is degraded in about 6 weeks. The growth factor facilitatedcell recruitments into the gel and promoted its degradation rate.

FIG. 15. Oscillatory time sweep measurements of LAPONITE® dispersiongelled with cationic and anionic molecules, (A) Lysine and (B) Glutamicacid. The LAPONITE® concentration is fixed at 19 mg/mL, while theconcentrations of lysine or glutamic acid are varied. (Diamond) 19+0.95mg/mL (20:1), (Triangle) 19+2.4 mg/mL (8:1) and (Circle) 19+4.8 mg/mL(4:1).

DETAILED DESCRIPTION

The use of numerical values in the various ranges specified in thisapplication, unless expressly indicated otherwise, are stated asapproximations as though the minimum and maximum values within thestated ranges are both preceded by the word “about”. In this manner,slight variations above and below the stated ranges can be used toachieve substantially the same results as values within the ranges.Also, unless indicated otherwise, the disclosure of these ranges isintended as a continuous range including every value between the minimumand maximum values. For definitions provided herein, those definitionsrefer to word forms, cognates and grammatical variants of those words orphrases. As used herein “a” and “an” refer to one or more.

As used herein, the term “patient” or “subject” refers to members of theanimal kingdom including but not limited to human beings and “mammal”refers to all mammals, including, but not limited to human beings.

As used herein, the “treatment” or “treating” of a wound or defect meansadministration to a patient by any suitable dosage regimen, procedureand/or administration route of a composition, device or structure withthe object of achieving a desirable clinical/medical end-point,including attracting progenitor cells, healing a wound, correcting adefect, etc.

As used herein, the terms “comprising,” “comprise” or “comprised,” andvariations thereof, are open ended and do not exclude the presence ofother elements not identified. In contrast, the term “consisting of” andvariations thereof is intended to be closed, and excludes additionalelements in anything but trace amounts.

By “biocompatible”, it is meant that a device, scaffold composition,etc. is essentially, practically (for its intended use) and/orsubstantially non-toxic, non-injurious or non-inhibiting ornon-inhibitory to cells, tissues, organs, and/or organ systems thatwould come into contact with the device, scaffold, composition, etc.

As used herein, the term “polymer composition” is a compositioncomprising one or more polymers. As a class, “polymers” includes,without limitation, homopolymers, heteropolymers, co-polymers, blockpolymers, block co-polymers and can be both natural and synthetic.Homopolymers contain one type of building block, or monomer, whereasco-polymers contain more than one type of monomer. An “oligomer” is apolymer that comprises a small number of monomers, such as, for example,from 3 to 100 monomer residues. As such, the term “polymer” includesoligomers. The term polypeptide includes polypeptides and oligopeptides.

A polymer “comprises” or is “derived from” a stated monomer if thatmonomer is incorporated into the polymer. Thus, the incorporated monomerthat the polymer comprises is not the same as the monomer prior toincorporation into a polymer, in that at the very least, certain linkinggroups are incorporated into the polymer backbone or are removed in thepolymerization process. A polymer is said to comprise a specific type oflinkage if that linkage is present in the polymer. An incorporatedmonomer is a “residue”.

A “hydrogel” is a water insoluble, three-dimensional network of polymerchains, and in the context of the present invention, silicate platelets,plus water that fills the voids between the polymer chains. Hydrogelsare mostly water (the mass fraction of water is much greater than thatof polymer). They do not flow, but they allow small molecules todiffuse, so long as those small molecules do not bind the platelets orpolymer. The hydrogels described herein are shear-thinning, meaning thatthey are non-Newtonian fluids that lose viscosity under shear strain,such as a stirring force, or forces encountered in a medical syringewhen the plunger is moved.

In one aspect a shear-thinning composition as described herein is usedas a carrier for release of a therapeutic agent e.g., for therapeuticpurposes. In one aspect, the therapeutic agent binds to a gelling agent,such as a sulfated or sulfamated polymer or an anionic or cationiccomposition, such as a polyanionic or polycationic oligopeptide. In oneaspect, the therapeutic agent binds specifically or non-specifically toa sulfated or sulfamated polymer. In one example, a therapeutic agent iscombined into the shear thinning composition, and the composition isthen implanted in a patient or otherwise administered to the patient,for example, by injection or application to a wound or another site in apatient suitable for delivery of the therapeutic agent. The therapeuticagent is added to the other ingredients in the shear-thinningcomposition by mixture with the gelling agent, such as a sulfated orsulfamated polymer, and the silicate platelets, or by adsorption to orabsorption into a gel formed by the gelling agent and the silicateplatelets. Generally, the therapeutic agents include any substance thatcan be mixed with, embedded into, absorbed into, adsorbed to, orotherwise attached to or incorporated onto or into the compositiondescribed herein or incorporated into a drug product that would providea therapeutic benefit to a patient. Non-limiting examples of suchtherapeutic agents include growth factors, cytokines, chemoattractants,antimicrobial agents, emollients, retinoids, antibodies and fragmentsthereof, and topical steroids. Each therapeutic agent may be used aloneor in combination with other therapeutic agents. For example and withoutlimitation, a composition comprising angiogenic agents may be applied toa wound.

In one aspect, a shear-thinning therapeutic composition is provided. Thecomposition comprises: silicate platelets, a gelling agent, such as asulfated or sulfamated polymer, non-covalently linking the platelets toform a shear-thinning composition; and a therapeutic agent thatnon-covalently binds to, specifically or non-specifically, the gellingagent and/or the platelets. For example, the therapeutic agent isnon-covalently bound to sulfated or sulfamated polymer complexednon-covalently with the silicate platelets to form a shear-thinning gel.

According to certain aspects, the ratio of therapeutic agent to thegelling agent, such as a sulfated or sulfamated polymer, e.g. heparin,ranges from 1:16000 to 1:1 by weight. In one aspect, the ratio oftherapeutic agent to the gelling agent, such as a sulfated or sulfamatedpolymer, e.g. heparin, ranges from 1:8000 to 1:3. In a further aspect,the ratio of therapeutic agent to the gelling agent, such as a sulfatedor sulfamated polymer, e.g., heparin, ranges from 1:1520 up to 1:3 byweight.

According to certain aspects, the ratio of the gelling agent, such as asulfated or sulfamated polymer, e.g., heparin, to the silicateplatelets, e.g. LAPONITE®, ranges from 1:10 to 1:1 by weight. Forexample, seen below, heparin-LAPONITE® gels are prepared from 1.9+19mg/mL to 19+19 mg/mL of heparin+LAPONITE®, resulting in rapid gelation.In one aspect, a ratio of sulfated or sulfamated polymer, e.g. heparinto LAPONITE® is 1.9+19 mg/mL to 9.5+19 mg/mL, or a sulfated orsulfamated polymer, e.g. heparin, to LAPONITE® ratio of from 1:10 to 1:2is used. Such H/L weight ratio is applicable to other combinations ofsulfated or sulfamated polymer with LAPONITE®, or other silicateplatelet compositions.

In aspects, the LAPONITE® concentration ranges from 10 mg/mL to 80 mg/mL(1% to 8%), and in certain aspects from 15 mg/mL to 50 mg/mL (1.5% to5.0%).

The above ratios of therapeutic agent to gelling agent, such as asulfated or sulfamated polymer, e.g. heparin, to silicate platelets arebased on studies of FGF2, heparin, and LAPONITE®. These results can begeneralized to other therapeutic agents, other gelling agent, such assulfated or sulfamated polymers, and other silicate platelets.

In the examples below, controlled release is demonstrated by loading 500ng FGF2 in 525 μL of heparin-LAPONITE® gels to formFGF2-heparin-LAPONITE® gels at 0.00095+3.8+19 mg/mL, 0.00095+7.6+19mg/mL and 0.00095+15.2+19 mg/mL. The FGF2 to heparin to LAPONITE® weightratios in these three formulations are 1:4000:20000, 1:8000:20000 and1:16000:20000. FGF2-heparin-LAPONITE® gel at 0.00095+7.6+19 mg/mL showedalmost 100 release in 34 days. Therefore, in one aspect, a protein toheparin ratio of 1:8000 is selected.

In the in vivo angiogenesis study below, 50 and 500 ng FGF2 was loadedin 100 μL of heparin-LAPONITE® gel to form FGF2-heparin-LAPONITE® gelsat 0.0005+7.6+19 mg/mL and 0.005+7.6+19 mg/mL. The subcutaneousimplantation with 500 ng FGF2-loaded heparin-LAPONITE® gel showed strongangiogenesis efficacy. Therefore in one aspect, a protein to heparinratio of 1:1520 used for an in vivo angiogenesis study in a mouse model,and in others aspect a protein to heparin ratio range from 1:1520 to 1:3by weight is provided.

As used herein, silicate platelets are found naturally in silica clays(natural platelets) or can be manufactured (synthetic platelets).Silicate clays are phyllosilicates (sheet silicates). In nature, thesheets often are microparticles (from 1 to 1000 microns in its largestcross-section) and/or nanoparticles (from 1 to 1000 nanometers in itslargest cross-section). Phyllosilicate clay particles are plate-likeparticles having a perimeter shape that is generally rounded (e.g.,circular, elliptical, or oval), polyhedral (e.g., hexagonal), or anyclosed figure, and are referred to as platelets (see, e.g. FIG. 1A).Platelets have an aspect ratio (the ratio of the largest dimension ofthe sheets/discs to thickness of the sheet/disk), for example andwithout limitation, of greater than 50, and more typically ranging from100 to 1,500. Silicate platelets typically comprise an oxide silicatewith alkali metals, alkaline earth metals, and/or hydroxide, optionallyincluding other elements, such as aluminum and iron.

In one aspect, the silicate platelets are LAPONITE®. LAPONITE® (hydroussodium lithium magnesium silicate) is a synthetic crystalline layeredsilicate colloid with crystal structure and composition closelyresembling the natural smectite clay hectorite. When dispersed in water,LAPONITE® hydrates and swells to form a clear colloidal dispersion withthe Na⁺ ions forming double layers on the faces. The pH for a 2%LAPONITE® suspension in pure water is ˜9.8. At low ionic strength,electrostatic repulsion keeps the particles apart. LAPONITE® isdecomposed by acids, leading to an increase in ion concentration withtime at low pH. At concentrations of 2% or greater in water a gel willform rapidly. LAPONITE® gel is strongly thixotropic, i.e. its viscositydecreases rapidly under shear. After the shear stress is removed, thegel reforms; the rate of restructuring depends on composition,electrolyte level, age of the dispersion, and temperature. The additionof salts reduces the thickness of the electrical double layer, promotinggel formation (Cummins H Z, Liquid, glass, gel: The phases of colloidalLAPONITE®, Journal of Non-Crystalline Solids 353 (2007) 3891-3905).

LAPONITE® XLG (Na⁺ _(0.7)[(Si₈Mg_(5.5)Li_(0.3))O₂₀(OH)₄]⁻ _(0.7)), asynthetic silicate, is a plate-like nanoparticle with negatively chargedsurface and positively charged edge (Cummins H Z., Journal ofNon-Crystalline Solids. 2007; 353:3891-905). LAPONITE® shows goodbiocompatibility and biodegrades into non-toxic and bioabsorbablebyproducts of Na⁺, Mg²⁺, Si(OH)₄ and Li⁺ (Gaharwar A K, et al.,Bioactive silicate nanoplatelets for osteogenic differentiation of humanmesenchymal stem cells. Adv Mater. 2013; 25:3329-36).

Gelling Agent

A gelling agent is used herein to non-covalently bind to or complex withthe silicate platelets in the shear-thinning composition.

In one aspect, the gelling agent is a sulfated or sulfamated polymer. Inone aspect, the sulfated or sulfamated polymer is a sulfated orsulfamated polysaccharide or oligosaccharide. Synthetic and naturalsulfated or sulfamated polysaccharides include oligosaccharides, andfurther include, for example and without limitation, sulfatedglycosaminoglycans or sulfated galactans, ulvans and fucans (See, e.g.,Jiao, G., et al. Chemical Structures and Bioactivities of SulfatedPolysaccharides from Marine Algae (2011) Mar. Drugs 9:196-223).Non-limiting examples of sulfated or sulfamated polysaccharides include,pentosan polysulfates, dermatan sulfates, keratan sulfates, chondroitinsulfates, sulfated agarans (e.g., porphyrans), and carageenans.

By “sulfated”, it is meant that the polymer comprises one or morependant sulfate (—OSO₃) groups. By “sulfamated” it is meant one or morependant sulfamate (—NSO₃) groups. By “sulfated or sulfamated” it ismeant a polymer comprises one or more sulfate groups, one or moresulfamate groups, or one or more each of sulfate groups and sulfamategroups. Examples of suitable sulfated or sulfamated polysaccharidesinclude, without limitation: a sulfated polysaccharide, a sulfamatedpolysaccharides, a sulfated or sulfamated polydisaccharide, a sulfatedglycosaminoglycan, heparin, and heparan sulfate (see, e.g., FIG. 2).

In another aspect, the sulfated or sulfamated polymer is a sulfated orsulfamated synthetic polymer, such as a polyurethane, polyester,polyurea, polyamide-ester, polyether, polycarbonate, polyamide, orpolyolefin, or copolymers thereof, as are broadly-known in the polymerarts. In another aspect, the sulfated or sulfamated polymer comprises atleast one moiety selected from the following:

-   -   (a)        [—OC(O)—CH(NHY)—CH₂—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂—]_(n),    -   (b)        [—OC(O)—CH₂—CH(NHY)—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂—]_(n),    -   (c)        [—OC(O)—CH(NHY)—CH₂—CH₂—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂—]_(n),        and/or    -   (d)        [—OC(O)—CH₂—CH₂—CH(NHY)—C(O)O—CH₂—CH(O—R1)-CH₂—O—CH₂—CH₂—O—CH₂—CH(O—R2)-CH₂—]_(n),        wherein Y is —C(O)—CH(NH₃ ⁺)—(CH₂)₃—NH—C(NH₂)₂ ⁺ or —C(O)—CH(NH₃        ⁺)—(CH₂)₄—(NH₃)⁺, n>1, and R1 and R2 are the same or different        and are independently selected from the group consisting of        sulfate-containing groups and sulfamate-containing groups, with        the composition having an overall negative charge. A        sulfate-containing group is a moiety (portion of a molecule)        comprising at least one pendant sulfate group. A        sulfamate-containing group is a moiety (portion of a molecule)        comprising at least one pendant sulfamate group.

In other aspects, the gelling agent is a cationic or anionic compound.Cationic and anionic molecules include the water soluble amino acidssuch as lysine, arginine, glutamic acid, aspartic acid, and hydrophilicamino acids, including glutamine, histidine, asparagine, serine,tyrosine, threonine and other water soluble amino acids; cationicoligo-peptides, such as the dimer, trimer, tetramer, pentamer, andhexamer etc. of the above mentioned cationic amino acids, optionallycomprising a plurality of the above mentioned cationic amino acids, suchas poly(arginine) or poly(lysine); anionic oligo-peptides andpolypeptides, e.g., of above mentioned anionic amino acids, optionallycomprising a plurality of the above mentioned anionic amino acids, suchas poly(glutamic acid) or poly(aspartic acid); and hydrophilicoligo-peptides and polypeptides, e.g., of above mentioned hydrophilicamino acids. In one aspect, oligomers of cationic amino acids are from 2to 10, or optionally from 2 to 6 amino acids. Oligomers or polypeptidesof anionic amino acid, or hydrophilic amino acids, or poly (anionicamino acids), or poly(hydrophilic amino acids), or combinations thereofare expected to have no limitation as to the number of amino acids in apeptide sequence.

Gelling agents according to any aspect described herein, may be modifiedto include biologically active groups or therapeutic agents covalentlybound (attached) to the polymer structure in addition to thenon-covalent inclusion of a therapeutic agent into the shear-thinningcomposition.

In preparation of the shear-thinning composition as described herein, atherapeutic agent is admixed with the gelling agent and silicateplatelets in any order that allows for formation of a shear-thinningcomposition. In one aspect, the therapeutic agent is first mixed withthe gelling agent, e.g., the sulfated or sulfamated polymer, accordingto any aspect described herein, and then is mixed with the silicateplatelets, according to any aspect described herein, to form ashear-thinning composition, e.g., a hydrogel. Therapeutic agents may becombined in safe and effective amounts. Therapeutic agents that arestrongly hydrophobic and/or non-polar might not combine directly to thesulfated or sulfamated polymer, but are expected to do so when combinedwith a pharmaceutically-acceptable excipient that is charged orotherwise compatible with the system as described herein, or whenmodified to include hydrophilic or charged groups—so long as thecomposition retains its pharmacological activity. In one aspect atherapeutic agent, e.g., a hydrophobic therapeutic agent or atherapeutic agent that is not otherwise compatible by itself with theshear thinning composition according to any aspect described herein, isfirst combined with a cyclodextrin, as are broadly-known in the drugdelivery arts.

Salt forms of many of therapeutic agents can be utilized, though thesalt counterion may need to be removed to avoid interfering withgelation of the composition described herein. Alternatively, the saltcounterion does not need to be removed, but the salt counterionconcentration should permit loading and gelling of the composition andformation of a shear-thinning composition. In one aspect, a combinationdosage form is provided that is a shear-thinning composition comprising:a gelling agent, e.g. a sulfated or sulfamated polymer, in any aspectdescribed herein; a first therapeutic agent that binds the gellingagent, e.g., the sulfated or sulfamated polymer, for example, where thesulfated or sulfamated polymer is heparin or heparan sulfate, the firsttherapeutic agent is a member of the heparin interactome; silicateplatelets, according to any aspect described herein; and a secondtherapeutic agent that is any therapeutic agent that does notsubstantially interfere with formation of the shear-thinningcomposition. In one aspect, the second therapeutic agent is anytherapeutic agent that does not interfere with the formation of ashear-thinning composition, such as a hydrogel. In another aspect, thesecond therapeutic agent does not bind to the gelling agent, e.g., thesulfated or sulfamated polymer. In yet another aspect, the secondtherapeutic agent binds specifically to the sulfated or sulfamatedpolymer. In a further aspect, the sulfated or sulfamated polymer isheparin or heparan sulfate, and the second therapeutic agent is a memberof the heparin interactome. Additional therapeutic agents may becombined in the composition, so long as the composition remainsshear-thinning.

Therapeutic agents that may be incorporated, by themselves, or incombination with a suitable excipient, into the compositions describedherein include, without limitation, anti-inflammatories, such as,without limitation, NSAIDs (non-steroidal anti-inflammatory drugs) suchas salicylic acid, indomethacin, sodium indomethacin trihydrate,salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal,diclofenac, indoprofen sodium salicylamide, antiinflammatory cytokines,and antiinflammatory proteins or steroidal anti-inflammatory agents);antibiotics; anticlotting factors such as heparin, Pebac, enoxaprin,aspirin, hirudin, plavix, bivalirudin, prasugrel, idraparinux, warfarin,coumadin, clopidogrel, PPACK, GGACK, tissue plasminogen activator,urokinase, and streptokinase; growth factors. Other therapeutic agentsinclude, without limitation: (1) immunosuppressants; glucocorticoidssuch as hydrocortisone, betamethisone, dexamethasone, flumethasone,isoflupredone, methylpred-nisolone, prednisone, prednisolone, andtriamcinolone acetonide; (2) antiangiogenics such as fluorouracil,paclitaxel, doxorubicin, cisplatin, methotrexate, cyclophosphamide,etoposide, pegaptanib, lucentis, tryptophanyl-tRNA synthetase, retaane,CA4P, AdPEDF, VEGF-TRAP-EYE, AG-103958, Avastin, JSM6427, TG100801,ATG3, OT-551, endostatin, thalidomide, becacizumab, neovastat; (3)antiproliferatives such as sirolimus, paclitaxel, perillyl alcohol,farnesyl transferase inhibitors, FPTIII, L744, antiproliferative factor,Van 10/4, 5-FU, Daunomycin, Mitomycin, dexamethasone, azathioprine,chlorambucil, methotrexate, mofetil, vasoactive intestinal polypeptide,and PACAP; (4) antibodies; (5) drugs acting on immunophilins, such ascyclosporine, zotarolimus, everolimus, tacrolimus and sirolimus(rapamycin), interferons, TNF binding proteins; (6) taxanes, such asdocetaxel; statins, such as atorvastatin, lovastatin, simvastatin,pravastatin, fluvastatin, and rosuvastatin; (7) nitric oxide donors orprecursors, such as, without limitation, Angeli's Salt, L-Arginine, FreeBase, Diethylamine NONOate, Diethylamine NONOate/AM, Glyco-SNAP-1,Glyco-SNAP-2, S-Nitroso-N-acetylpenicillamine, S-Nitrosoglutathione,NOC-5, NOC-7, NOC-9, NOC-12, NOC-18, NOR-1, NOR-3, SIN-1, SodiumNitroprusside, Dihydrate, Spermine NONOate, Streptozotocin; and (8)antibiotics, such as, without limitation: acyclovir, afloxacin,ampicillin, amphotericin B, atovaquone, azithromycin, ciprofloxacin,clarithromycin, clindamycin, clofazimine, dapsone, diclazaril,doxycycline, erythromycin, ethambutol, fluconazole, fluoroquinolones,foscarnet, ganciclovir, gentamicin, iatroconazole, isoniazid,ketoconazole, levofloxacin, lincomycin, miconazole, neomycin,norfloxacin, ofloxacin, paromomycin, penicillin, pentamidine, polymixinB, pyrazinamide, pyrimethamine, rifabutin, rifampin, sparfloxacin,streptomycin, sulfadiazine, tetracycline, tobramycin, trifluorouridine,trimethoprim sulphate, Zn-pyrithione, ciprofloxacin, norfloxacin,afloxacin, levofloxacin, gentamicin, tobramycin, neomycin, erythromycin,trimethoprim sulphate, polymixin B, and silver salts such as chloride,bromide, iodide, and periodate.

Any useful cytokine or chemoattractant can be mixed into, mixed with, orotherwise combined with any composition as described herein. For exampleand without limitation, useful components include growth factors,interferons, interleukins, chemokines, monokines, hormones, andangiogenic factors. In certain non-limiting aspects, the therapeuticagent is a growth factor, such as a neurotrophic or angiogenic factor,which optionally may be prepared using recombinant techniques.Non-limiting examples of growth factors include basic fibroblast growthfactor (bFGF), acidic fibroblast growth factor (aFGF), vascularendothelial growth factor (VEGF), hepatocyte growth factor (HGF),insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), platelet derivedgrowth factor (PDGF), stromal derived factor 1 alpha (SDF-1 alpha),nerve growth factor (NGF), ciliary neurotrophic factor (CNTF),neurotrophin-3, neurotrophin-4, neurotrophin-5, pleiotrophin protein(neurite growth-promoting factor 1), midkine protein (neuritegrowth-promoting factor 2), brain-derived neurotrophic factor (BDNF),tumor angiogenesis factor (TAF), corticotrophin releasing factor (CRF),transforming growth factors α and β (TGF-α and TGF-β), interleukin-8(IL-8), granulocyte-macrophage colony stimulating factor (GM-CSF),interleukins, and interferons. Commercial preparations of various growthfactors, including neurotrophic and angiogenic factors, are availablefrom R & D Systems, Minneapolis, Minn.; Biovision, Inc, Mountain View,Calif.; ProSpec-Tany TechnoGene Ltd., Rehovot, Israel; and CellSciences®, Canton, Mass.

Non-limiting examples of angiogenic therapeutic agents include:erythropoietin (EPO), basic fibroblast growth factor (bFGF), acidicfibroblast growth factor (aFGF), fibroblast growth factor-2 (FGF-2),granulocyte colony stimulating factor (G-CSF), granulocyte macrophagecolony stimulating factor (GM-CSF), hepatocyte growth factor (HGF),insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), placental growthfactor (PIGF), platelet derived growth factor (PDGF), stromal derivedfactor 1 alpha (SDF-1 alpha), vascular endothelial growth factor (VEGF),angiopoietins (Ang 1 and Ang 2), matrix metalloproteinase (MMP),delta-like ligand 4 (D114), and class 3 semaphorins (SEMA3s), all ofwhich are broadly-known, and are available from commercial sources.

In certain non-limiting aspects, the therapeutic agent is anantimicrobial agent, such as, without limitation, isoniazid, ethambutol,pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones,ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin,dapsone, tetracycline, erythromycin, ciprofloxacin, doxycycline,ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine,sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone,paromomycin, diclazaril, acyclovir, trifluorouridine, foscarnet,penicillin, gentamicin, ganciclovir, iatroconazole, miconazole,Zn-pyrithione, and silver salts such as chloride, bromide, iodide, andperiodate.

In certain non-limiting aspects, the therapeutic agent is ananti-inflammatory agent, such as, without limitation, an NSAID, such assalicylic acid, indomethacin, sodium indomethacin trihydrate,salicylamide, naproxen, colchicine, fenoprofen, sulindac, diflunisal,diclofenac, indoprofen, sodium salicylamide; an anti-inflammatorycytokine; an anti-inflammatory protein; a steroidal anti-inflammatoryagent; or an anti-clotting agents, such as heparin. Other drugs that maypromote wound healing and/or tissue regeneration may also be included.

Non-limiting examples of antiangiogenic agents include: Macugen(pegaptanib sodium); Lucentis; Tryptophanyl-tRNA synthetase (TrpRS);AdPEDF; VEGF TRAP-EYE; AG-013958; Avastin (bevacizumab); JSM6427;TG100801; ATG3; Perceiva (originally sirolimus or rapamycin); E10030,ARC1905 and colociximab (Ophthotech) and Endostatin. Ranibizumab iscurrently the standard in the United States for treatment of neovascularAMD. It binds and inhibits all isoforms of VEGF. Although effective inmany cases, treatment with ranibizumab requires sustained treatmentregimens and frequent intravitreal injections. VEGF Trap is a receptordecoy that targets VEGF with higher affinity than ranibizumab and othercurrently available anti-VEGF agents. Blocking of VEGF effects byinhibition of the tyrosine kinase cascade downstream from the VEGFreceptor also shows promise, and includes such therapies as vatalanib,TG100801, pazopanib, AG013958 and AL39324. Small interfering RNAtechnology-based therapies have been designed to downregulate theproduction of VEGF (bevasiranib) or VEGF receptors (AGN211745). Otherpotential therapies include pigment epithelium-derived factor-basedtherapies, nicotinic acetylcholine receptor antagonists, integrinantagonists and sirolimus. (See, e.g., Chappelow, A V, et al.,Neovascular age-related macular degeneration: potential therapies,Drugs. 2008; 68(8):1029-36 and Barakat M R, et al., VEGF inhibitors forthe treatment of neovascular age-related macular degeneration, ExpertOpin Investig Drugs. 2009 May; 18(5):637-46.)

In another aspect, antioxidants are added to the polymeric composition,such as organic or inorganic antioxidants. In one aspect, theantioxidant is a nanoparticle incorporated by any means into the polymercomposition, such as, for example, a cerium nanoparticle. As an example,an anisotropic heart valve or heart valve leaflet prosthesis ismanufactured by electrospinning, or by any useful method, and ceriumnanoparticles are deposited in and/or on the prosthesis either during orafter manufacture.

In one aspect, the therapeutic agent is a member of the interactome ofthe sulfated or sulfamated polymer that optionally directly binds to, oris a ligand of, the sulfated or sulfamated polymer. By “interactome”(e.g., interaction network) it is meant proteins that interact, howevertransiently, with a specified composition, and includes both specificand non-specific binding of the therapeutic agent to the sulfated orsulfamated polymer. In one aspect, the member of the specifiedinteractome binds to, and optionally directly binds to the specifiedcomposition, e.g. sulfated or sulfamated polymer, and therefore can becharacterized as a ligand of the specified composition. The interactomesof various sulfated or sulfamated polymers or oligomers are described inthe art. For example, the glycosaminoglycan-protein interaction networkis described in Gesslbauer, B., et al., (Exploring theGlycosaminoglycan-Protein Interaction Network by Glycan-MediatedPull-Down Proteomics, Electrophoresis 2016 June; 37(11):1437-47). Ori,A, et al., (A Systems Biology Approach for the Investigation of theHeparin/Heparan Sulfate Interactome, J. Biol. Chem. Jun. 3, 2011;286(22):19892-19904) describe the heparin/heparan sulfate interactome.For example, Ori, A, et al. describes 442 members of the heparin/heparansulfate interactome, including, for example and without limitation, thefollowing and cleavage products, recombinant versions, mutated versions,and/or post-translationally modified versions analogs or derivativesthereof that non-covalently bind heparin/heparan sulfate:

4F2 cell-surface antigen heavy chain; 5′-nucleotidase;Alpha-1-antitrypsin; Alpha-1B-glycoprotein; Alpha-2-macroglobulin;Amyloid beta A4 protein; Soluble APP-alpha; Soluble APP-beta; C99;Beta-amyloid protein 42; Beta-amyloid protein 40; C83; P3(42); P3(40);C80; Gamma-secretase C-terminal fragment 59; Gamma-secretase C-terminalfragment 57; Gamma-secretase C-terminal fragment 50; C31;Alpha-1-antichymotrypsin; Angio-associated migratory cell protein; Bilesalt export pump; ATP-binding cassette sub-family G member 2;ATP-binding cassette sub-family G member 5; Amiloride-sensitive amineoxidase; Alpha-1B adrenergic receptor; Agouti-related protein; AminoacyltRNA synthase complex-interacting multifunctional protein 1; Aldosereductase; Protein AMBP; Inter-alpha-trypsin inhibitor light chain;Trypstatin; Alpha-2-macroglobulin receptor-associated protein;Angiogenin; Angiotensinogen; Angiotensin-2; Angiotensin-3;Antithrombin-III; Annexin A1; Annexin A2; Annexin A3; Annexin A5;Annexin A6; Amyloid-like protein 1; Amyloid-like protein 2;Apolipoprotein A-V; Apolipoprotein B-100; Apolipoprotein E;Beta-2-glycoprotein 1; Aquaporin-1; Arginase-1; Artemin;Agouti-signaling protein; Sodium/potassium-transporting ATPase subunitalpha-1; Sodium/potassium-transporting ATPase subunit beta-1;Sodium/potassium-transporting ATPase subunit beta-3; Plasma membranecalcium-transporting ATPase 1; Copper-transporting ATPase 2; ATPsynthase subunit alpha mitochondrial; Attractin; A disintegrin andmetalloproteinase with thrombospondin motifs 1; A disintegrin andmetalloproteinase with thrombospondin motifs 3; A disintegrin andmetalloproteinase with thrombospondin motifs 5; A disintegrin andmetalloproteinase with thrombospondin motifs 8; A disintegrin andmetalloproteinase with thrombospondin motifs 9; Beta-2-microglobulin;Band 3 anion transport protein; cDNA FLJ57339; Beta-secretase 1; Bonemorphogenetic protein 2; Bone morphogenetic protein 3; Bonemorphogenetic protein 4; Bone morphogenetic protein 7; Probetacellulin;Complement C1q subcomponent subunit A; Complement C1q subcomponentsubunit B; Complement C1q subcomponent subunit C; C4b-binding proteinalpha chain; Voltage-dependent L-type calcium channel subunit alpha-1S;Cadherin-8; Azurocidin; Cathepsin B; Cathepsin B heavy chain]; CathepsinG; Corticosteroid-binding globulin; Carboxypeptidase B2;Carboxypeptidase D; Coiled-coil domain-containing protein 134;Coiled-coil domain-containing protein 80; C—C motif chemokine 1;Eotaxin; C—C motif chemokine 13; C—C motif chemokine 13 medium chain;C—C motif chemokine 13 short chain; C—C motif chemokine 15;CCL15(25-92); CCL15(29-92); C—C motif chemokine 17; C—C motif chemokine19; C—C motif chemokine 2; C—C motif chemokine 21; C—C motif chemokine22; MDC(5-69); MDC(7-69)]; C—C motif chemokine 23; CCL23(22-99);CCL23(27-99); CCL23(30-99)]; C—C motif chemokine 24; C—C motif chemokine25; C—C motif chemokine 27; C—C motif chemokine 28; C—C motif chemokine3; C—C motif chemokine 4; C—C motif chemokine 5; RANTES; C—C motifchemokine 7; C—C motif chemokine 8; Fibronectin type-IIIdomain-containing protein C4orf31; Antigen-presenting glycoprotein CD1d;Platelet glycoprotein 4; Leukocyte surface antigen CD47; Bilesalt-activated lipase; Ceruloplasmin; Uncharacterized protein C6orf15;Complement factor B; Complement factor B Bb fragment; Complement factorD; Complement factor H; Complement factor 1; Complement factor 1 lightchain; Chordin; UPF0765 protein C10orf58; Clusterin; Clusterin alphachain; Chymase; Collagen alpha-1; Collagen alpha-2(I) chain; ComplementC2; Complement C2a fragment; Collagen alpha-1(II) chain; Complement C3;Complement C3 alpha chain; C3a anaphylatoxin; Complement C3b alpha′chain; Complement C3c alpha′ chain fragment 1; Complement C3dg fragment;Complement C3g fragment; Complement C3d fragment; Complement C3ffragment; Complement C3c alpha′ chain fragment 2; Collagen alpha-1(III)chain; Complement C4-A; Complement C4-A alpha chain; C4a anaphylatoxin;C4b-A; C4d-A; Complement C4 gamma chain]; Collagen alpha-1(IV) chain;Collagen alpha-2(IV) chain; Complement C5; Complement C5 alpha chain;C5a anaphylatoxin; Complement C5 alpha′ chain; Collagen alpha-1(V)chain; Collagen alpha-3(V) chain; Complement component C6; Collagenalpha-3(VI) chain; Complement component C7; Complement component C8alpha chain; Complement component C8 beta chain; Complement component C8gamma chain; Complement component C9; Complement component C9b; Collagenalpha-1(IX) chain; Collagen alpha-1(XI) chain; Collagen alpha-2(XI)chain; Collagen alpha-1(XII) chain; Collagen alpha-1(XIII) chain;Collagen alpha-1(XIV) chain; Collagen alpha-1(XVIII) chain; Collagenalpha-1(XIX) chain; Acetylcholinesterase collagenic tail peptide;Cartilage oligomeric matrix protein; Catechol O-methyltransferase;Collagen alpha-1(XXIII) chain; Collagen alpha-1(XXV) chain; Calciumrelease-activated calcium channel protein 1; Cysteine-rich secretoryprotein LCCL domain-containing 2; Granulocyte-macrophagecolony-stimulating factor; Connective tissue growth factor; Low affinitycationic amino acid transporter 2; Gap junction beta-1 protein; C—X—Cmotif chemokine 2; C—X—C motif chemokine 6; Small-inducible cytokine B6N-processed variant 2; Small-inducible cytokine B6 N-processed variant3]; Platelet basic protein; TC-2; Connective tissue-activating peptideIII; Beta-thromboglobulin; Neutrophil-activating peptide 2(74);Neutrophil-activating peptide 2(73); Neutrophil-activating peptide 2;TC-1; Neutrophil-activating peptide 2(1-66); Neutrophil-activatingpeptide 2(1-63); C—X—C motif chemokine 10; C—X—C motif chemokine 11;C—X—C motif chemokine 13; Cytochrome c; Protein CYR61; Netrin receptorDCC; Estradiol 17-beta-dehydrogenase 11; Estradiol 17-beta-dehydrogenase12; 17-beta-hydroxysteroid dehydrogenase 13; 3-keto-steroid reductase;Dipeptidyl peptidase 4; Dipeptidyl peptidase 4 soluble form;Endothelin-converting enzyme 1; Extracellular matrix protein 2;Ephrin-A1; Ephrin-A3; Ephrin-A5; Elastin; Neutrophil elastase;Alpha-enolase; Ectonucleotide pyrophosphatase/phosphodiesterase familymember 1; Nucleotide pyrophosphatase (NPPase); Ectonucleotidepyrophosphatase/phosphodiesterase family member 3; Nucleotidepyrophosphatase; Receptor tyrosine-protein kinase erbB-2; Coagulationfactor X; Factor X heavy chain; Activated factor Xa heavy chain;Coagulation factor XI; Coagulation factor XIa light chain; Coagulationfactor XII; Beta-factor XIIa part 1; Beta-factor XIIa part 2;Coagulation factor XIIa light chain; Protein FAM55A; Coagulation factorIX; Coagulation factor IXa heavy chain; Fibulin-7; Fibrillin-1;Fibrillin-2; Fibrosin-1; IgG receptor FcRn large subunit p51; Fetuin-B;Heparin-binding growth factor 1; Fibroblast growth factor 10; Fibroblastgrowth factor 12; Fibroblast growth factor 14; Fibroblast growth factor16; Fibroblast growth factor 17; Fibroblast growth factor 18;Heparin-binding growth factor 2; Fibroblast growth factor 20; Fibroblastgrowth factor 22; Fibroblast growth factor 3; Fibroblast growth factor4; Fibroblast growth factor 5; Fibroblast growth factor 6; Keratinocytegrowth factor; Fibroblast growth factor 8; Glia-activating factor;Fibroblast growth factor-binding protein 1; Fibroblast growthfactor-binding protein 3; Basic fibroblast growth factor receptor 1;Fibroblast growth factor receptor 2; Fibroblast growth factor receptor3; Fibroblast growth factor receptor 4; Fibrinogen alpha chain;Fibrinogen beta chain; Fibrinogen gamma chain; Fibronectin; Ugl-Y1;Ugl-Y2; Ugl-Y3; Follistatin; Follistatin-related protein 1; Furin;Protein G6b; Glia-derived nexin; Glial cell line-derived neurotrophicfactor; Gelsolin; Growth hormone receptor; G-protein coupled receptor182; Transmembrane glycoprotein NMB; Growth-regulated alpha protein;GRO-alpha(5-73); GRO-alpha(6-73); Solute carrier family 2; facilitatedglucose transporter member 2; Proheparin-binding EGF-like growth factor;Hepatoma-derived growth factor; Heparin cofactor 2; Hereditaryhemochromatosis protein; Hepatocyte growth factor; Hepatocyte growthfactor beta chain; High mobility group protein B1; Haptoglobin;Haptoglobin beta chain; Histidine-rich glycoprotein; Islet amyloidpolypeptide; Insulin-like growth factor-binding protein 2; Insulin-likegrowth factor-binding protein 3; Insulin-like growth factor-bindingprotein 4; Insulin-like growth factor-binding protein 5; Insulin-likegrowth factor-binding protein 6; Plasma protease C1 inhibitor;Interferon gamma; Indian hedgehog protein Indian hedgehog proteinC-product; Interferon-inducible GTPase 5; Interleukin-10; Interleukin-12subunit beta; Interleukin-2; Interleukin-3; Interleukin-4;Interleukin-5; Interleukin-6; Interleukin-7; Interleukin-8;Interleukin-8; IL-8(5-77); IL-8(6-77); IL-8(7-77); IL-8(8-77);IL-8(9-77); Interphotoreceptor matrix proteoglycan 2; Inhibin beta Achain; Insulin receptor; Insulin receptor subunit beta; Plasma serineprotease inhibitor; Integrin alpha-1; Integrin alpha-5; Integrin alpha-5light chain; Integrin alpha-M; Integrin alpha-V; Integrin alpha-V lightchain; Integrin beta-1; Integrin beta-3; Inter-alpha-trypsin inhibitorheavy chain H3; Integral membrane protein 2B; Anosmin-1; Putativekeratinocyte growth factor-like protein 1; Putative keratinocyte growthfactor-like protein 2; Kininogen-1; T-kinin; Bradykinin;Lysyl-bradykinin; Kininogen-1 light chain; Low molecular weightgrowth-promoting factor]; Laminin subunit alpha-1; Laminin subunitalpha-2; Laminin subunit alpha-3; Laminin subunit alpha-4; Lamininsubunit alpha-5; Laminin subunit gamma-2; Leucyl-cystinylaminopeptidase; Low-density lipoprotein receptor; Galectin-9;Leucine-rich repeat-containing G-protein coupled receptor 4; Leukemiainhibitory factor receptor; Hepatic triacylglycerol lipase; Endotheliallipase; Lipoprotein lipase; Platelet-activating factor acetylhydrolaseIB subunit alpha; Latrophilin-2; Latent-transforming growth factorbeta-binding protein 1; L-selectin; P-selectin; Mannose-binding proteinC; Multidrug resistance protein 1; Multidrug resistance protein 3;Hepatocyte growth factor receptor; Macrophage migration inhibitoryfactor; Midkine; Matrix metalloproteinase-14; 72 kDa type IVcollagenase; Matrilysin; Matrix metalloproteinase-9; 82 kDa matrixmetalloproteinase-9; Monocarboxylate transporter 1; Monocarboxylatetransporter 8; Multidrug resistance-associated protein 6; Myosinregulatory light polypeptide 9; Neuron navigator 2; Neural cell adhesionmolecule 1; Netrin-1; Nicastrin; Noggin; Pro-neuregulin-1;membrane-bound isoform; Neuropilin-1; Neurturin; Sodium/bile acidcotransporter; Occludin; Zinc finger protein OZF; Calcium-dependentphospholipase A2; Phospholipase A2; membrane associated; Plasminogenactivator inhibitor 1; Plasminogen activator inhibitor 1 RNA-bindingprotein; Proton-coupled folate transporter; Procollagen C-endopeptidaseenhancer 2; Proprotein convertase subtilisin/kexin type 5; Proproteinconvertase subtilisin/kexin type 6; Programmed cell death protein 5;Platelet-derived growth factor subunit A; Platelet-derived growth factorsubunit B; Protein disulfide-isomerase; Protein disulfide-isomerase A6;Phosphatidylethanolamine-binding protein 1; Platelet endothelial celladhesion molecule; Pigment epithelium-derived factor; Myeloperoxidase;84 kDa myeloperoxidase; Myeloperoxidase light chain; Myeloperoxidaseheavy chain; Platelet factor 4 variant; Platelet factor 4 variant(5-74);Platelet factor 4 variant(6-74); Basement membrane-specific heparansulfate proteoglycan core protein; LG3 peptide; Biglycan; Polymericimmunoglobulin receptor; Putative phospholipase B-like 1; Plateletfactor 4; Placenta growth factor; Plasminogen; Activation peptide;Angiostatin; Plasmin heavy chain A short form; Plasmin light chain B;Serum paraoxonase/arylesterase 1; Serum paraoxonase/arylesterase 2;Serum paraoxonase/lactonase 3; Periostin; Peptidyl-prolyl cis-transisomerase B; Peroxiredoxin-4; Prolargin; Bone marrow proteoglycan; Majorprion protein; Prolactin; Vitamin K-dependent protein C; VitaminK-dependent protein C heavy chain; Activation peptide; Properdin;Presenilin-1; Presenilin-1 CTF subunit; Presenilin-1 CTF12; Proteinpatched homolog 1; Pleiotrophin; Receptor-type tyrosine-proteinphosphatase C; Stromal cell-derived factor 1 gamma; Liver-specificorganic anion transporter 3TM13; SLCO1A2 protein; Mannan-bindingprotein; 60S ribosomal protein L22; 60S ribosomal protein L29;Roundabout homolog 1; R-spondin-1; R-spondin-2; R-spondin-3;R-spondin-4; 40S ribosomal protein SA; Solute carrier family 12 member9; Sodium-dependent phosphate transporter 2; Solute carrier family 22member 1; Solute carrier family 22 member 7; Solute carrier family 22member 18; Sodium-coupled neutral amino acid transporter 3;Sodium-coupled neutral amino acid transporter 4; Zinc transporter ZIP4;Electrogenic sodium bicarbonate cotransporter 1; Serum amyloid Aprotein; Serum amyloid protein A(2-104); Serum amyloid protein A(3-104);Serum amyloid protein A(2-103); Serum amyloid protein A(2-102); Serumamyloid protein A(4-101); Serum amyloid P-component; Sodium channelprotein type 5 subunit alpha; Stromal cell-derived factor 1;SDF-1-alpha(3-67); Semaphorin-5A; Semaphorin-5B; Secretedfrizzled-related protein 1; Sonic hedgehog protein; Sonic hedgehogprotein C-product; Beta-galactoside alpha-2,6-sialyltransferase 1; Slithomolog 1 protein; Slit homolog 2 protein; Antileukoproteinase;Synaptogyrin-1; Superoxide dismutase [Cu—Zn]; Extracellular superoxidedismutase [Cu—Zn]; Sortilin; Sclerostin; Stabilin-2; EGF-like,laminin-type, EGF-like and link domain-containing scavenger receptor 2;Metalloreductase STEAP4; Stromal interaction molecule 1;Alpha-synuclein; Microtubule-associated protein tau; Teneurin-1;Tenascin; Tenascin-X; Tissue factor pathway inhibitor; Transferrinreceptor protein 1, serum form; Transferrin receptor protein 2;Transforming growth factor beta receptor type 3; Transforming growthfactor beta-1; Transforming growth factor beta-2; Protein-glutaminegamma-glutamyltransferase 2; Thioredoxin; Prothrombin; Thrombin lightchain; Thrombin heavy chain; Thyroglobulin; Metalloproteinase inhibitor3; T-cell immunomodulatory protein; Tumor necrosis factor ligandsuperfamily member 13; Tumor necrosis factor; Tumor necrosis factorsoluble form; Tissue-type plasminogen activator; Tissue-type plasminogenactivator chain B; Tumor necrosis factor receptor superfamily member11B; Serotransferrin; Lactotransferrin; Lactoferroxin-A;Lactoferroxin-B; Lactoferroxin-C; Trypsin-1; Tryptase alpha/beta-1;Tryptase beta-2; Tumor necrosis factor-inducible gene 6 protein;Thrombospondin-1; Thrombospondin-2; Thrombospondin-3; Thrombospondin-4;Transthyretin; Urokinase-type plasminogen activator; Vascularendothelial growth factor A; Vascular endothelial growth factor B;Vascular endothelial growth factor receptor 1; Vascular endothelialgrowth factor receptor 2; Vitamin D-binding protein (DBP) (VDB)(Gc-globulin) (Group-specific component); Vitronectin; von Willebrandfactor; Proto-oncogene Wnt-1; Fractalkine; Lymphotactin; Xanthinedehydrogenase/oxidase; Zinc transporter 1 (ZnT-1); and ProteinZ-dependent protease inhibitor.

Pharmaceutically acceptable salts of any therapeutic agent bound to orotherwise combined with, or incorporated into the shear-thinningcomposition according to any aspect herein, may be employed.Pharmaceutically acceptable salts are, because their solubility in wateris greater than that of the initial or basic compounds, particularlysuitable for medical applications. These salts have a pharmaceuticallyacceptable anion or cation. Suitable pharmaceutically acceptable acidaddition salts of the compounds of the invention include, withoutlimitation, salts of inorganic acids such as hydrochloric acid,hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acid, andof organic acids such as, for example, acetic acid, benzenesulfonic,benzoic, citric, ethanesulfonic, fumaric, gluconic, glycolic,isethionic, lactic, lactobionic, maleic, malic, methanesulfonic,succinic, p-toluenesulfonic, and tartaric acid. Suitablepharmaceutically acceptable basic salts include without limitation,ammonium salts, alkali metal salts (such as sodium and potassium salts),alkaline earth metal salts (such as magnesium and calcium salts), andsalts of trometamol (2-amino-2-hydroxymethyl-1,3-propanediol),diethanolamine, lysine, or ethylenediamine. Pharmaceutically acceptablesalts may be prepared from parent compounds by any useful method, as arewell known in the chemistry and pharmaceutical arts. It should be notedthat for inorganic salts, the content to be loaded in this hydrogel maybe limited, because addition of salts will promote gelation of thesilicate platelets. As such, in certain instances, salt forms may not beused, or counterions should be removed, for example by ion exchange,prior to mixing the therapeutic agent or therapeutic agent plus sulfatedor sulfamated polymer with the silicate platelets. However, some saltcontent, for example as shown in FIG. 7, will actually promote gelation.

As used herein, the terms “drug” and “drugs” refer to any compositionshaving a preventative or therapeutic effect, including and withoutlimitation, antibiotics, peptides, hormones, organic molecules,vitamins, supplements, factors, proteins and chemoattractants. A“therapeutic agent” (e.g., FGF-2, below) is the pharmacologically-activeconstituent of a drug product (e.g., the shear thinning compositionbelow comprising LAPONITE®, heparin, and FGF-2, also referred to hereinas a therapeutic composition.

According to one aspect of the invention, a method of making ashear-thinning therapeutic composition (a drug product) is provided. Themethod comprises mixing a sulfated or sulfamated polymer with atherapeutic agent that is a binding partner of the sulfated polymers oroligomers to produce a complex of the sulfated or sulfamated polymer andthe therapeutic agent; and mixing the complex of the sulfated orsulfamated polymer and the therapeutic agent with natural or syntheticsilicate platelets to produce a shear-thinning hydrogel. According toadditional aspects, one or more additional, different, therapeuticagent(s) are combined with the sulfated or sulfamated polymers oroligomers and/or the natural or synthetic silicate platelets prior to orduring any mixing step to produce a combined dosage form. Alternatively,the therapeutic agent is first mixed with the silicate platelets andthen with the sulfated or sulfamated polymer. In yet another aspect, thetherapeutic agent is mixed with the silicate platelets and with thesulfated or sulfamated polymer at the same time.

In use, the shear-thinning composition according to any aspect describedherein is administered to a patient. For example, the composition isadministered parenterally, e.g., subcutaneously or intramuscularly. Inone aspect, the composition is placed into or is distributed in amedical syringe. During delivery, a force is applied, resulting in aloss of viscosity of the composition, and the composition is passedthrough a needle, cannula, catheter, or any useful tube for delivery ofthe composition at a site for delivery in a patient, for example andwithout limitation, within a wound, abdomen or peritoneal cavity of apatient. Small globules of the composition can be placed at any suitablesite of the patient, or within a tissue engineering scaffold. In oneaspect, the composition is injected or otherwise implanted at a site ofa wound or tissue defect in the patient, wherein the composition is usedas a soft tissue filler, which can find use in plastic surgery andreconstructive surgery, for example in the case of maxillofacial injuryor defect repair, or in breast reconstructive surgery.

In a further aspect, a commercial kit is provided comprising acomposition described herein. A kit comprises suitable packagingmaterial and the shear-thinning composition according to any aspectdescribed herein. In one non-limiting aspect, the kit comprises atherapeutic shear-thinning composition according to any aspect describedherein in a vessel, which is the packaging, or which is contained withinpackaging. In various aspects, the vessel is a vial, a medical syringe,tube or any other container suitable for storage and transfer incommercial distribution routes of the kit.

Example 1

A versatile hydrogel for tissue regeneration that preserves thebioactivity of growth factors is tested. The shear-thinning gelself-assembles within one minute from heparin and LAPONITE®. By notcovalently modifying heparin, it retains its natural affinity towardsmany proteins anchored in the extracellular matrix. In principle,Heparin-LAPONITE® gel can bind any heparin-binding compound orcomposition, e.g., a growth factor. Fibroblast growth factor-2 (FGF2) isused as proof-of-concept. Heparin in the gel protects FGF2 fromproteolytic degradation and allows it to be released over time withpreserved bioactivity. FGF2 released from subcutaneously injected geland induces strong angiogenesis in a mouse model. The hydrogel degradescompletely in vivo in 8 weeks with or without growth factors, elicitingmild inflammatory response but having little impact on the surroundingtissue. The ease of preparation and scale-up makes this protein deliveryplatform attractive for clinical translation.

Materials and Methods:

Preparation of Heparin-LAPONITE® hydrogel (Heparin-LAPONITE®): LAPONITE®XLG and heparin sodium salt were donated by BYK Additives Inc. (Texas,USA) and Scientific Protein Laboratories, LLC (Waunakee, USA)respectively. All LAPONITE® dispersions were prepared by dispersingLAPONITE® powder in deionized water with vigorously magnetic stirringfor 2 h before use. Concentration of heparin solutions ranged from 40 to400 mg/mL and were prepared by dissolving heparin sodium salt indeionized water. When preparing Heparin-LAPONITE® gels, 0.1 mL ofheparin solution was quickly added to 2.0 mL of LAPONITE® dispersion andimmediately manually swirled for one minute to yield Heparin-LAPONITE®gel. In this way, 0.1 mL of 40 to 400 mg/mL heparin solutions wereseparately mixed with 2.0 mL of 20 mg/mL LAPONITE® to yieldheparin-LAPONITE® gels comprised of heparin and LAPONITE® withconcentrations ranged from 1.9+19 to 19+19 mg/mL, respectively. Theweight ratio of heparin to LAPONITE® (H/L) of the as-madeHeparin-LAPONITE® gels was ranged from 1:10 to 10:10.

To prepare Heparin-LAPONITE® gels at different solid concentrations withH/L ratio fixed at 4:10, 40 mg of LAPONITE® powder was dispersed incertain volumes of deionized water to obtain LAPONITE® dispersions withconcentrations ranged from 15 to 30 mg/mL, respectively. Then eachdispersion was mixed with 0.1 mL of 160 mg/mL heparin solution to yieldHeparin-LAPONITE® gels with solid concentrations (heparin+LAPONITE®)ranged from 5.8+14.4 to 11.2+28.0 mg/mL. All heparin-LAPONITE® gels wereprepared and immediately used for rheological tests.

To prepare heparin-LAPONITE® gels containing different content of NaClwith H/L ratio fixed at 4:10, 0.1 mL of heparin solutions containingNaCl ranged from 2.1 to 18.9 wt. % were dropwisely added to 2.0 mL of 20mg/mL LAPONITE® dispersion while maintaining manually swirling duringaddition. The mixtures were immediately transferred for rheologicaltests. In this way, the Heparin-LAPONITE® gels containing 0.1 to 0.9 wt.% NaCl were prepared to examine the effects of salt ions on the gelationkinetics and gel properties.

Rheological study: Dynamic rheological measurements were performed onAR2000ex (TA Instruments, USA). The as-made Heparin-LAPONITE® gels withdifferent H/L ratio or solid concentrations were immediately transferredto a parallel plate (40 mm diameter, gap distance 750 μm) for rheometrytest. To prevent evaporation of solvent, a thin layer of mineral oil wasapplied to the sample edge during test. Oscillatory time sweepmeasurement was performed to record the gelation behavior versus time at37° C. under a controlled strain of 1% and a frequency of 1 rad s⁻¹. Toinvestigate viscoelastic properties, angular frequency sweep measurementwas conducted to record both storage (G′) and loss (G″) moduli withangular frequency setup from 0.1 to 100 rad s⁻¹ and a strain at 1%.Furthermore, a steady shear rate sweep was performed to investigate theshear thinning behavior of the hydrogels as a function of shear ratefrom 0.01 to 10 s⁻¹. Each sample was replicated at least three times.

Zeta potential test: Zeta potential measurements were performed usingzetasizer (Nano-ZS90, Malvern). The LAPONITE® dispersion was diluted to10 mg/mL and heparin solutions were prepared with concentrations rangingfrom 20 to 160 mg/mL for use. Then 0.1 mL of 20 to 160 mg/mL heparinsolutions was separately mixed with 2.0 mL of 10 mg/mL LAPONITE®dispersion to yield a series of Heparin-LAPONITE® complex suspensions.These Heparin-LAPONITE® complex suspensions were comprised of heparinand LAPONITE® with concentrations between 0.95+9.5 to 7.6+9.5 mg/mL(H/L, 1:10 to 8:10). The Heparin-LAPONITE® complexes were prepared underthis condition to avoid gelation, so that the mobility of the conductivecomponents would not be hindered for zeta potential measurements. TheH/L ratio of the Heparin-LAPONITE® complexes was remained same to theHeparin-LAPONITE®gels. A LAPONITE® dispersion at 9.5 mg/mL and a seriesof heparin solutions with concentrations between 0.95 to 7.6 mg/mL werealso prepared for comparison. The freshly made Heparin-LAPONITE® complexsuspensions, LAPONITE® dispersion and heparin solutions were immediatelytransferred into a cuvette for zeta potential measurements. At leastthree readings of each measurement were recorded to calculate the meanvalue and its standard deviation.

Controlled release study: For in vitro and in vivo studies, allLAPONITE® dispersion and heparin in deionized water solutions weresterilized by filtering through 0.2 μm syringe filter before use. 500 ngFGF2 was loaded in Heparin-LAPONITE® gels with different heparin toLAPONITE® ratio (H/L) for controlled release study. Specifically, 2 μLof 250 ng/μL FGF2 solution was separately mixed with 25 μL of 80, 160and 320 mg/mL heparin solutions to yield three heparin-FGF2 complexes.The three heparin-FGF2 complexes were then separately added to 500 μL of20 mg/mL LAPONITE® dispersion in each Eppendorf tube and manuallyswirled for 1 min to yield three 500 ng FGF2-loaded Heparin-LAPONITE®gels. The three Heparin-LAPONITE® gels were comprised of heparin andLAPONITE® with concentrations of 3.8+19, 7.6+19 and 15.2+19 mg/mL (H/Lratio, 1:5, 2:5 and 4:5) to examine the H/L ratio effects on the FGF2release kinetics. The FGF2-loaded Heparin-LAPONITE® gels werecentrifuged at 5000 rpm for 15 second to remove air bubbles and then 200μL of 0.9% NaCl saline solution was added to the gel top. The controlledrelease was set up at 37° C. in an incubator for 34 days. At eachscheduled time point, the gel tubes were centrifuged at 5000 rpm for 15second and the supernatant was collected using pipette, and then 200 μLof fresh saline was added for next time point. Such procedure wasrepeated until the controlled release experiment was completed. Thecontrolled release was replicated three times.

500 ng FGF2 was directly loaded in two LAPONITE® gels with concentrationat 22.8 and 26.6 as controls to compare the release behavior fromHeparin-LAPONITE® gels. 2 μL of 250 ng/μL FGF2 solution was mixed with25 μL deionized water and then respectively added to 500 μL of 24 and 28mg/mL LAPONITE® dispersions and manually swirled for 1 min. TheFGF2-loaded LAPONITE® dispersions were incubated at 37° C. for about 2 hto yield two LAPONITE® gels containing 500 ng FGF2 in each gel. Allother procedure is same with FGF2-loaded Heparin-LAPONITE® gels forcontrolled release study. The concentrations of all the released FGF2samples were determined using ELISA assay kit according to themanufacturer's instruction (PEPROTECH, NJ, USA).

Western blot assay: To insure enough amount of the released FGF2 forWestern blot test, 5 μg FGF2 was loaded in Heparin-LAPONITE®, hyaluronicacid-LAPONITE® (HA-LAPONITE®) gels and LAPONITE® control, respectively.Specifically, 5 μL of 1 μg/μL FGF2 was mixed with 25 μL of 160 mg/mLheparin solution and then added to 500 μL of 20 mg/mL LAPONITE®dispersion with manually swirling for 1 min to yield 5 μg FGF2-loadedHeparin-LAPONITE® gel. Same amount of free FGF2 solution was dilutedwith 25 μL of deionized water and then directly mixed with 500 μL of 20mg/mL LAPONITE® dispersion to yield 5 μg FGF2-loaded LAPONITE® gel as acontrol.

HA-LAPONITE® gel was also prepared using a reported protocol (DivyaBhatnagar DX, Dilip Gersappe, and Miriam H. Rafailovich. Hyaluronic Acidand Gelatin Clay Composite. Journal of Chemical and BiologicalInterfaces. 2014; 2:1-11) for controlled release of FGF2 to compare withour Heparin-LAPONITE® gel. 5 μL of 1 μg/μL FGF2 solution was mixed with100 μL of 5 mg/mL hyaluronic acid and then added to 500 μL of 30 mg/mLLAPONITE® dispersion with manually swirling for 1 min to yield 5 μgFGF2-loaded HA-LAPONITE® gel.

All FGF2-loaded hydrogels were centrifuged at 5000 rpm for 15 second toremove any air bubbles and 500 μL fresh saline solution was added toeach gel top. The controlled release was set up at 37° C. in anincubator for 134 h. The gel tubes were centrifuged at 5000 rpm for 15second and the supernatants were collected. The concentrations of thereleased FGF2 were determined by ELISA assay to be 490 ng/mL forHeparin-LAPONITE® gel, and ca. 9.0 and 9.6 ng/mL for both LAPONITE®control and HA-LAPONITE® gel.

According to the released FGF2 concentration, free FGF2 solution at 490ng/mL and heparin-FGF2 complex solution at 3.9 mg/mL+490 ng/mL wereprepared as controls. Then 100 μL of the released FGF2 solution, freeFGF2 and heparin-FGF2 complex were separately mixed with 9.8 μg trypsin(FGF2:trypsin=1:200 by wt.) and incubated at 37° C. for 0.5 and 2 h,respectively. The digested solutions were immediately mixed with 100 μLof tricine sample buffer (Bio-Rad Laboratories, CA, USA) and denaturedat 100° C. for 5 min. Similarly, FGF2 solutions released from LAPONITE®and HA-LAPONITE® gels were similarly treated with trypsin and denaturedwith the tricine sample buffer using same procedure.

Western blotting was used to examine the amount of intact FGF2. SDS-PAGEwas utilized for separation followed by protein blotting on a PVDFmembrane (Bio-Rad Laboratories, Hercules, Calif.). The membrane wasblocked with 5% BSA in TBS with 0.05% Tween 20 for 1 h, then incubatedwith a rabbit anti-human FGF2 polyclonal antibody (1:1,000, Abcam,Cambridge, Mass.). The membranes were washed with TBS 3 times andincubated with a peroxidase conjugated anti-rabbit IgG antibody for 2 hat room temperature. Signals were visualized using the ChemiDic™XRS+Imaging System (Bio-Rad Laboratories, Hercules, Calif.), and banddensities were quantified using NIH ImageJ software.

In Vivo Biocompatibility and Biodegradation: Male BALB/cJ mice (JacksonLaboratory) with an average age of 8-9 wk were used and cared for incompliance with a protocol approved by the Institutional Animal Care andUse Committee of the University of Pittsburgh. Under isofluraneanesthesia, 100 μL of Heparin-LAPONITE® gel (7.6+19 mg/mL) or LAPONITE®gel (19 mg/mL) was injected in the left back of the mice through a 31 Ginsulin needle. The right back, which did not receive injection, servedas the contralateral control. All groups contained four to eight mice.The animals were sacrificed at post-injection day 3, weeks 2, 4, 6, and8. The subcutaneous tissues were harvested at the injection site and thecontralateral site. Tissues were fixed in 10% formalin for 15 min, andthen soaked in 30% sucrose and embedded in the Tissue-Tek optimumcutting temperature (O.C.T) compound (Sakura Finetek USA). Crosssections (6-μm thick, longitudinal axial cut) were stained withhematoxylin and eosin (H & E), and Masson's trichrome stain (MTS) andED-1 to examine inflammation or any adverse effects. For ED-1immunohistochemical analysis, 6-μm thick sections of the tissues weredried and fixed in histology-grade absolute ethanol for 15 min, airdried, and incubated with rat monoclonal anti-CD68 (1:200, Abcam,Cambridge, Mass.) for ED-1 identification. The slides were thenincubated with a goat anti-rat-Alexa 594 (1:400, Life technologies,Carlsbad, Calif.) for 1 hour. ED-1 stained sections were analyzed forthe density of newly recruited macrophages. Five to ten 200×magnification images were obtained for each specimen. The image wastaken using an inverted microscope Eclipse Ti (Nikon, Melville, N.Y.)equipped with a digital camera (QImaging, BC, Canada).

Angiogenesis Study: 100 μL of Heparin-LAPONITE® gel with or withoutloading growth factor FGF2 (500 ng) (Peprotech, Rocky Hill, N.J.) wasinjected in the left back of the mice through a 31 G insulin needle. Thesubcutaneous tissues were harvested on day 3, weeks 2 and 4post-injection and embedded and frozen in Tissue-Tek OCT compound.Sections of 6-μm thickness were prepared with a cryomicrotome. Thefollowing antibodies were applied per supplier instructions: ratanti-mouse CD31 monoclonal antibody (BD Biosciences), a goatanti-rat-Alexa 594, FITC-conjugated anti-α-SMA monoclonal antibody(Sigma). All slides were counterstained with DAPI (Invitrogen). Thefluorescent images were taken by a Nikon inverted microscope Eclipse Ti.Six low magnification (100×) fields containing the highest number ofCD31- or α-SMA-positive cells were randomly selected for each group. Thenumber of blood vessels in the field was counted and confirmed byDAPI-positive nuclei. The value was divided by the area of the imagedtissue to obtain blood vessel number per unit area and a mean value wascalculated based on the six images per group.

Statistical Analysis: The data of macrophage number and blood vesselnumber per square millimeter were analyzed using one-way ANOVAstatistical analysis with post-hoc Bonferroni correction. A p value<0.05 is considered significant. Data represent mean±standard deviation(SD).

Results and Discussion

Rapid gelation between heparin and LAPONITE®: Heparin-LAPONITE® gel isdesigned for protein delivery by mixing heparin-protein complex solutionwith LAPONITE® dispersion and manually swirled to gel within one minute(FIG. 3A). To examine the gelation rate between the components, 20 mg/mLof LAPONITE® dispersion was selected to mix with heparin solution toyield Heparin-LAPONITE® gel with H/L ratio ranged from 1:10 to 10:10(1.9+19 mg/mL to 19+19 mg/mL). The as-made LAPONITE® dispersion itselfat this concentration cannot form a weak gel through face-to-edgeinteraction within about 69 h (Table 1). Dynamic rheologicalmeasurements on gelation kinetics, viscoelastic properties revealed theshear thinning behavior of the Heparin-LAPONITE® gel. Oscillatory timesweep measurements indicate that the storage (G′) and loss (G″) moduliare proportional to the heparin content when the LAPONITE® concentrationis fixed at 19 mg/mL (FIG. 3B). The first data points of the measured G′and G″ from time sweep measurements were plotted as a function ofheparin concentration to evaluate the gelation rate upon mixing the twocomponents (FIG. 3D). It clearly shows that the G′ and G″ values quicklyincrease from approximately 0.01 to 120 Pa and 0 to 10 Pa respectivelywhen heparin concentration increases from 1.9 to 5.7 mg/mL (H/L, 1:10 to3:10), followed by a slow elevation to 150 and 12 Pa at heparinconcentration of 9.5 mg/mL (H/L, 5:10). While further increasing heparincontent leads to gradually reduced G′ and G″ again (FIG. 3D, 9.5 to 19mg/mL). Angular frequency sweep measurements demonstrate that theHeparin-LAPONITE® gels all have stable G′ values approximately 7-16times higher than G″ values throughout the frequency sweep from 0.1 to100 rad s⁻¹ except the sample formed at low H/L ratio of 1:10 (FIG. 3C).These results indicate that the heparin and LAPONITE® can quickly form astable network structure within the appropriate H/L ratio range (asabove, in one aspect, a useful H/L weight ratio range is between 1:10 to1:1 with a LAPONITE® concentration between 10 to 80 mg/mL, andoptionally between 15-50 mg/mL, and in this example, 20 mg/mL LAPONITE®was chosen to examine the quick gelation for H/L ratio ranged from 1:10to 1:1 by weight). The viscosity of LAPONITE® dispersion was highlyrelated to stirring time in deionized water (exfoliating degree). 50mg/mL LAPONITE® mixed with heparin was used to form a gel and it couldbe taken into syringe for injection before it became solid-like gel.Generally, below 35 mg/mL LAPONITE® is dispersed in deionized water withvigorous stirring for more than 2 h for use; while above 35 mg/mL, itcan be stirred for a short time for use, e.g., about 10-60 min accordingto its concentration. Such network shows more elastic than viscousproperties, which is not disrupted under suitable shear stress due tothe rapid reversible interactions. A shear rate sweep measurement wasperformed to confirm the shear thinning behavior of the gel forinjectability (FIG. 3E). Based on these investigations, we speculatethat the rapid gelation and stable gel properties can be attributed toseveral aspects: (1) the negatively charged heparin can diffuse into theLAPONITE® dispersion without microscopic aggregation; (2) theelectrostatic interactions between the two components form quickly andthus establish reversible ionic crosslinks rapidly; and (3) the stableLAPONITE® dispersion with uniform particle size facilitates uniformgelation.

TABLE 1 Gelation time of LAPONITE ® dispersion at differentconcentration. LAPONITE ®, mg/mL 20 26.6 32.9 39.1 Gelation time* 69 h20 min 7 min 1 min *The LAPONITE ® dispersions were prepared and thensettled for gelation. Gelation time was recorded by inverting each vialfor at least 30 seconds without observing any flowable gel.

The LAPONITE® nanoplatelet carries approximately 1000 negative chargesper particle and the particle edge is positively charged which countersapproximately 10% of the total negative charges, the rest of thenegative charges are balanced by metal ions. The heparin used here hasnumber average molecular weight of ˜20 kDa and each chain bearsapproximately 117 negative charges, though it is believed that a largemolecular weight range for heparin or heparan sulfate would be useful(FIG. 2). Owing to such high negative charge density, when suitableamount of the heparin solution is added to LAPONITE® dispersion, heparinmolecules can easily diffuse between the nanoplatelets under only gentleswirling for a minute with no need of mechanical stirring to formhomogenous mixture. It is theorized that most of the heparin chains arequickly anchored onto LAPONITE® edges, forming effective crosslinks viaelectrostatic interactions, instead of absorbing on LAPONITE® surfacesdue to the strong negative charge repulsion (FIG. 3A). Because of thelimited heparin chain length and contact areas between the particleedges, it is likely that only some of the anchored heparin can bridgethe adjacent LAPONITE® edges to establish effective crosslinks. This iswhy the rheometry measurements show that the starting G′ upon mixingdepends highly on heparin content (FIG. 3D, 1.9 to 5.7 mg/mL). Then aslow elevation to the maximal G′ value at 9.5 mg/mL (H/L, 5:10)indicates that an optimal H/L ratio is reached for quickest gelation. Inthe following region (9.5 to 19 mg/mL), though more free heparins existin the system, it is estimated that most of them are just anchored ontoLAPONITE® edges as pendent chains with little contribution tocrosslinking. It appears that the LAPONITE® edges are gradually maskedby absorbing more free heparin molecules when the heparin concentrationexceeds 9.5 mg/mL, leading to a reduction of the areas for bridging bythe anchored chains thus hindering the gelation process. Such phenomenonwas observed in PEO gelling with LAPONITE® (Mongondry P, et al.,Influence of pyrophosphate or polyethylene oxide on the aggregation andgelation of aqueous LAPONITE® dispersions. Journal of colloid andinterface science. 2004; 275:191-6).

It is expected that the gelation rate is proportional to LAPONITE®concentration because the spaces between the particles are reduced athigher concentration and thus the anchored heparins can more effectivelyreach the adjacent particles to form crosslinks. This is consistent withoscillatory rheological measurements of Heparin-LAPONITE® gels as afunction of the concentration with the H/L ratio fixed at 2:5 (FIG. 4).It shows that both G′ and G″ are proportional to the concentrations, butthe maximal modulus is reached at 36.1 mg/mL, indicating thatexcessively high concentration of LAPONITE® likely hinders heparindiffusion and retard gelation. Here, the H/L weight ratio is fixed at2:5. Namely the gel is comprised of 10.3 mg/mL heparin+25.8 mg/mLLAPONITE®. In this case, the LAPONITE® concentration is ranged from 15mg/mL to 30 mg/mL, which is mixed with heparin at fixed ratio of 2:5(H/L ratio) to test their viscoelastic properties versus LAPONITE®concentrations. In all the range from 15 mg/mL to 50 mg/mL, heparin canquickly complex with LAPONITE® to form gel with H/L weight ratio rangedbetween 1:10 to 1:1.

LAPONITE® dispersion at 20 mg/mL is stable for at least 6 months andonly forms weak “house-of-cards” gel after mature gelation (FIG. 5A).The weak gel can be easily converted into low-viscosity dispersion byvigorously shaking for approximately 2 minutes, which can then bere-gelled quickly upon addition of heparin if desired (FIG. 5B). This isimportant for practical use of the gel because it avoids preparation offresh dispersion every time. As a drug delivery system, the gel willcontact with physiological fluid after injection. This will potentiallyaffect the viscoelastic properties, which might in turn influence therelease kinetics of the payloads. Thus the gel stability and propertieswere further examined by incubating a freshly made heparin-LAPONITE® gelwith excessive biological saline and cell medium (EBM-2, Lonza,Walkersville, Md.). The angular frequency sweep measurements indicatethat little impacts are caused on the viscoelastic properties of the gelby either saline or EBM-2 over different incubation time (FIG. 6A, B).In addition, pH condition on the gelation behavior is also examinedusing LAPONITE® dispersion prepared in deionized water with pH levelsranged from 2.8 to 10.6. When mixing with heparin, all LAPONITE®dispersions show similar gelation behavior and viscoelastic propertiesas confirmed by oscillatory time sweep measurements (FIG. 6C).Furthermore, the effects of ionic strength on gelation kinetics werealso investigated to examine if the presence of salt ions will inhibitor retard gelation process.

The heparin was dissolved in NaCl solution and then added dropwise toLAPONITE® dispersion to yield heparin-LAPONITE® gels (7.6+19 mg/mL)containing 0.1 to 0.9 wt. % NaCl. The oscillatory time sweepmeasurements show that the presence of suitable amount of salt ionspromoted the gelation process, instead of inhibiting or retardinggelation (FIG. 7). The G′ and G″ are increased gradually and almosttripled as the NaCl content increases from 0 to 0.9 wt. %. This isbecause the addition of salts reduces the thickness of the electricaldouble layer on the LAPONITE® surfaces, promoting gel formation. In thiscase, the anionic heparin together with the NaCl salts facilitatedgelation kinetics with increased moduli at the tested content range. Allthese studies further confirmed the robust gelation ability betweenheparin and LAPONITE® to yield a stable gel for applications.

Zeta potential to examine the electrostatic interactions: We believethat electrostatic interactions crosslink heparin and LAPONITE®, thuszeta potential was used to examine the change of charge upon mixing thetwo components (FIG. 8). In this case, the LAPONITE® concentration was9.5 mg/mL, so that it can complex heparin without forming a gel. Thelatter will hinder the mobility of conductive components for zetapotential measurement. The heparin-LAPONITE® complex suspensions haveconcentrations (heparin+LAPONITE®) ranged from 0.95+9.5 to 7.6+9.5 mg/mLand the H/L ratio remained same to the heparin-LAPONITE® gels, rangedfrom 1:10 to 4:5. Compare to the zeta potential of heparin and LAPONITE®control, the Heparin-LAPONITE® complexes show little increase of zetapotential at heparin concentrations between 0.95 to 4.75 mg/mL (H/L,1:10 to 1:2), followed by a sharp increase between 4.75 to 5.7 mg/mL(H/L, 1:2 to 3:5) and then a plateau between 5.7 to 7.6 mg/mL (H/L, 3:5to 4:5). Such zeta potential profile indicates that most of thenegatively charged heparin molecules are bound to the positively chargedLAPONITE® edges at H/L ratio between 1:10 to 5:10. Few free heparinscontribute to the zeta potential values at this range. The sharpincrease of ZP implies a saturating absorption of heparin to LAPONITE®occurred at H/L ratio between 1:2 to 3:5. Above this threshold, morefree heparin molecules contribute to zeta potential value in a similartrend as the heparin solution control shows (FIG. 8). Here, thesaturating absorption occurs at H/L ratio between 1:2 to 3:5, which isslightly higher than that determined by rheological study at 1:2 (FIG.3D). In that case of heparin-LAPONITE® gel, the two components quicklyform network structure, and some of the anchored heparin chains areshared by the adjacent particles to form crosslinks. Thus it shows aslightly lower H/L ratio for the saturating absorption compared to theheparin-LAPONITE® complex. Nonetheless, the zeta potential measurementprovides a good evidence for the proposed crosslinking mechanism basedon electrostatic interactions between heparin and LAPONITE® edges.

Controlled release of protein and protection from proteolyticdegradation: Most free growth factors degrade quickly in vivo byproteolytic cleavage, leading to low therapeutic efficacy. Theadvantages of the heparin-LAPONITE® delivery platform are the protectionagainst proteolysis and sustained release of the proteins in an activeform. This is demonstrated using FGF2 as a model biomolecule toinvestigate its release kinetics and stability with regard toproteolytic degradation (FIG. 9).

The FGF2 is loaded in heparin-LAPONITE® gels with H/L ratios of 1:5, 2:5and 4:5. The H/L ratio is varied to compare the effects on its releasekinetics. Same amount of free FGF2 is directly loaded in LAPONITE® gelswith concentrations at 22.8 and 26.6 mg/mL as controls. The releaseprofiles show that the cumulative release is elevated from 25.3±4.7% to105±11% over 34 days when the H/L ratio increases from 1:5 to 2:5, butis reduced to 22.6±0.2% again when the H/L ratio is further increased to4:5. However, both LAPONITE® controls show negligible FGF2 release(0.8±0.2 and 1.3±0.3%) over 34 days. Such release kinetics is mainlydependent on the binding between FGF2, heparin and LAPONITE®. InHeparin-LAPONITE® gels, FGF2 is complexed with heparin to formheparin-binding proteins and then incorporated in the gels (FIG. 3A).When at low H/L ratio of 1:5, the heparin-FGF2 complex is more easilyabsorbed onto LAPONITE® edges via electrostatic interaction, showing aslower and steadier release compared to the one with the H/L ratio at2:5. Contrary to initial expectations, the gel with higher H/L ratio at4:5 results in a slower release again, instead of faster release. It wassuspected that this is mainly due to more free heparin moleculesinteracting with FGF2 to retain the latter within the gel, thusdecreasing the release rate. When the free FGF2 is loaded in theLAPONITE® controls, the FGF2 molecules are directly bound to thenanoplatelets through hydrogen bonding, electrostatic interaction andphysical absorption. The strong bounds fix the protein in the gelleading to very low levels of release.

The ability of heparin-LAPONITE® gel to protect FGF2 against proteolysisis examined by Western blot (FIG. 9B, C). The FGF2 released fromHeparin-LAPONITE® gel over 134 h at 37° C. was collected for Westernblot assay, which was compared to free FGF2 and heparin-FGF2 complexcontrols. After incubating with protease (trypsin) for 0.5 and 2 h, theFGF2 released from the Heparin-LAPONITE® gel (490 ng/mL determined byELISA assay), showing band intensity similar to the heparin-FGF2 complexcontrol, while the free FGF2 was completely digested by trypsintreatment within 0.5 h (FIG. 9B). This result demonstrates effectiveprotection of the FGF2 from protease degradation and indicates that thereleased FGF2 is probably bound to heparin, rather than in the freeform. The FGF2 released from LAPONITE® control and hyaluronicacid-LAPONITE® gel (HA-LAPONITE®) is also examined for comparison withHeparin-LAPONITE® gel (FIG. 9C). HA-LAPONITE® is prepared as arepresentative example to compare to the Heparin-LAPONITE® gel forprotein delivery (Divya Bhatnagar D X, et al., Hyaluronic Acid andGelatin Clay Composite. Journal of Chemical and Biological Interfaces.2014; 2:1-11). Neither LAPONITE® nor HA-LAPONITE® gel could yielddetectable FGF2 even before trypsin treatment. This is mainly due tovery low release of FGF2 from both LAPONITE® and HA-LAPONITE® gels asdetermined by ELISA assay (˜9.0 and 9.6 ng/mL FGF2 released, 0.090% and0.096% cumulative release over 134 h). This result implies that FGF2 inHA-LAPONITE® gel is released in a similar way as compared to theLAPONITE® control. This is possibly because most, if not all, of theproteins bind directly to the LAPONITE® nanoplatelets because thehyaluronic acid has low affinity for FGF2, leading to extremely lowdissociation of FGF2 from HA-LAPONITE® gel and likely no protection fromenzymatic degradation as well. The comparison among Heparin-LAPONITE®,LAPONITE® and HA-LAPONITE® gels further demonstrated the versatility ofHeparin-LAPONITE® gel for controlled release of bioactive proteins.

In vivo biocompatibility study: Heparin-LAPONITE® gel with an H/L ratioat 2:5 (7.6+19 mg/mL) was selected as a representative for subcutaneousimplantation study to investigate the host response. All animalssurvived throughout the study without any malignancy, infection orabscess observed at the injection sites. Tissues around the gel showedno adverse reactions such as necrosis, fibrosis and muscle degeneration(FIG. 10A). H&E staining showed phagocytic inflammatory infiltrates nearthe gel at day 3 post-injection (FIG. 10A(a, d)). The inflammatoryinfiltrates reduce significantly at week 2 post-injection and phagocyticcells migrate into and proliferate inside the gel (FIG. 10A(b, e)). Mostof the macrophages migrated into the gel at week 4 (FIG. 10A(c, f)). Thesurrounding tissue was spared from inflammation throughout the studyperiod. MTS staining shows a relatively loose layer of collagen andminimal deposition on the surface of the gel at week 2 and 4post-injection (FIG. 10B). It is known that the inflammatory response toimplanted biomaterials activates the immune cells (e.g. macrophages) toinitiate the production of inflammatory cytokines and chemokines. Thesesecreted peptides typically recruit more immune cells to theimplantation site. Here, the recruited cells appear to accumulate on geledge and then migrate into the gel.

To further investigate cell activities, CD68 and DAPI were used todetect macrophages distribution relative to the subcutaneous implants.CD68 is a pan-macrophage marker. By day 3 after injection, the tissueadjacent to the gel contained most inflammatory cells, as revealed byCD68 staining. This is attributed to a non-specific inflammatoryresponse to the material, which has been widely observed in manyimplanted biomaterials. At week 2, there was a significant reduction innewly-recruited macrophages compared to day 3 (FIG. 11A, B), indicatingthat inflammation close to the surface of the gel was resolved at week2. On the other hand, the inflammatory cells inside the gel increased,but not significantly, at week 4 post-injection (FIG. 11B, C, D). Mostlikely the gel degraded sufficiently to allow cell infiltration deeperinto the gel.

FGF2-loaded Heparin-LAPONITE® gel induces strong angiogenesis: In orderto examine the bioactivity of the proteins released from the gel, FGF2was chosen as a model drug, and angiogenesis as a readout of FGF2activity. Therapeutic angiogenesis is a potential treatment for manyhuman diseases such as coronary and peripheral ischemia (Chu H, et al.,Proc. Nat'l. Acad. Sci. U.S.A. 2011; 108:13444-9 and Gupta R, et al.Human studies of angiogenic gene therapy. Circ. Res. 2009; 105:724-36).The FGF2-loaded Heparin-LAPONITE® gel (Heparin-LAPONITE®-FGF2) andHeparin-LAPONITE® alone were subcutaneously injected in the back ofBALB/cJ mice. The tissues surrounding the implantation site and thecontralateral side without gel treatment were harvested at differenttime points to study the effects of the released FGF2 on angiogenesis.The mature blood vessel formation was confirmed by co-staining of CD31(endothelial cell marker) and α-smooth cell actin (SMA, a mural cellmarker). Compared to the heparin-LAPONITE® alone treated group and thecontralateral control, the heparin-LAPONITE®-FGF2 gel-treated groupgenerated more mature blood vessels with larger diameter (FIG. 12A-C).Whereas the heparin-LAPONITE® alone group showed no significantdifference compared to its contralateral control (FIG. 12A, B). Theseresults indicate that the released FGF2 from heparin-LAPONITE®-FGF2 gelplays a significant role in promoting the formation of mature bloodvessels. To further compare the blood vessel number induced byheparin-LAPONITE®-FGF2 gel, heparin-LAPONITE® alone and thecontralateral control, the blood vessel number per square millimeter wasquantified by randomly selecting 10-20 images surrounding theimplantation site (FIG. 12D). The number of mature blood vessels perunit area induced by heparin-LAPONITE®-FGF2 gel was more than 3 times tothat of the contralateral control. Compare to the contralateral control,the heparin-LAPONITE® alone treatment cannot induce angiogenesis at 2weeks post-injection. Furthermore, prior studies showed that free FGF2treatment only demonstrated a limited angiogenesis due to the shorthalf-life in vivo (FIG. 12D) (Chu H, et al., Proc. Nat'l. Acad. Sci.U.S.A. 2011; 108:13444-9). The significantly enhanced angiogenesisindicates a well preserved bioactivity of the released FGF2 fromheparin-LAPONITE® gel.

The FGF2-loaded Heparin-LAPONITE® gel shows faster in vivo degradationthan heparin-LAPONITE® alone because the cells are more quicklyrecruited into the gel (FIG. 13). The heparin-LAPONITE®-FGF2 gel wasnearly completely degraded in 6 weeks after injection, about 2 weeksfaster than heparin-LAPONITE® alone (FIG. 14). The accelerated cellrecruitments and gel degradation should facilitate the applications ofthis gel because this study indicates that angiogenesis can be achievedwithin 2 weeks and fast gel degradation after that would minimizeforeign body response.

In sum, a versatile injectable hydrogel was designed for bioactiveprotein delivery with stabilized molecular structure and preservedbioactivity for tissue regeneration. This gel is made from heparinsolution and LAPONITE® dispersion by simply mixing the two components atappropriate ratio by manually swirling for one minute. FGF2 is used asan example for in vitro and in vivo studies to investigate its releasekinetics, stability against proteolytic degradation and bioactivity forangiogenesis. This delivery system is versatile and is expected to beuseful for protection and therapeutic delivery of many other proteins aswell. More than 400 proteins and peptides bind with heparin.

Example 2

This example illustrates the robust gelation ability between LAPONITE®and ionic amino acid molecules, cationic lysine and anionic glutamicacid. The easy gelation using various cationic, anionic and hydrophilicmolecules with LAPONITE® is expected to expand the applications asversatile drug delivery vehicles because different cargos can choosedifferent cationic, or anionic, or hydrophilic molecules to complex andincorporate into the gel for sustainable release.

Here, small biomolecules such as cationic and anionic amino acids areused to further examine the rapid gelation with LAPONITE® dispersion.Two types of amino acids are used as the representatives of smallmolecular gelators. One is cationic lysine (K) and the other is anionicglutamic acid (E). The K is readily dissolved in deionized water to formsolutions with concentrations ranged from 20 to 100 mg/mL for use. E isless soluble in pure deionized water, so it requires using 0.13 M and0.65 M NaOH solution to dissolve the amino acid to yield clear Esolutions (20 and 100 mg/mL) for use.

0.1 mL of K or E solution was added to 2 mL of LAPONITE® dispersion (20mg/mL) and manually swirled for couple of seconds to form LAPONITE®-Kand LAPONITE®-E mixtures. The mixtures were immediately transferred to aparallel plate for oscillatory rheometry tests. FIG. 15 shows thegelation kinetics of LAPONITE®-K and LAPONITE®-E as a functional oftime. It shows that both E and K can quickly gel with LAPONITE®dispersion. It is believed that K (cationic amino acid) moleculesinteract with negative charges on LAPONITE® platelet faces to promotegelation; while E (anionic amino acid) molecules interact with positivecharges on LAPONITE® edge to facilitate gelation process. An exemplaryweight ratio range of LAPONITE®-K for gelation is between 20:1 to 4:1,and the gelation rate is proportional to the K fraction. Beyond theratio of 4:1, LAPONITE®-K forms cloudy viscous mixture with a reducedstorage modulus. For LAPONITE®-E, the weight ratio can be increasedabove 4:1, but further increase of E fraction will not contribute toeither gelation rate or storage modulus.

LAPONITE® was reported as carriers for delivery of amino acids (M.Ghadiri, et al., Layered silicate clay functionalized with amino acids:wound healing application, RSC Advances, 2014, 4, 35332), but theseexperiments revealed that these ionic molecules can be employed asefficient gelators (gelling agents) to gel with LAPONITE® via ionicinteractions. In addition to lysine and glutamic acid, as therepresentatives of cationic and anionic molecules to demonstrate thequick gelation with LAPONITE®, other ionic amino acids such as arginineand aspartic acid, and hydrophilic amino acids, including glutamine,histidine, asparagine, serine, tyrosine, threonine and other watersoluble amino acids; cationic oligopeptides such as the dimer, trimer,tetramer, pentamer, and hexamer, etc. of the above mentioned cationicamino acids; anionic oligo-peptides and polypeptide of above mentionedanionic amino acids; and hydrophilic oligo-peptides and polypeptides ofabove mentioned hydrophilic amino acids. Above, heparin is shown as arepresentative of sulfated or sulfamated polymers to gel with LAPONITE®.Together, all these shear thinning hydrogels formed between ionicbiomolecules and LAPONITE® can expand the versatility of this techniquefor drug delivery applications.

The following clauses illustrate various aspects of the invention:

-   1. A shear-thinning therapeutic composition, comprising:    -   a. silicate platelets;    -   b. a gelling agent non-covalently linking the platelets to form        a shear-thinning composition; and    -   c. a therapeutic agent that binds non-covalently to the gelling        agent and that is complexed non-covalently with the gelling        agent.-   2. The composition of clause 1, wherein the gelling agent is a    sulfated or sulfamated polymer.-   3. The composition of clause 2, wherein the sulfated or sulfamated    polymer is a sulfated glycosaminoglycan.-   4. The composition of clause 3, wherein the sulfated    glycosaminoglycan is heparin or heparan sulfate.-   5. The composition of any one of clauses 1-4, wherein the    therapeutic agent is a member of the heparin interactome.-   6. The composition of clause 2, wherein the sulfated or sulfamated    polymer is a polysaccharide.-   7. The composition of any one of clauses 2-5, wherein the    therapeutic agent binds specifically to the sulfated or sulfamated    polymer.-   8. The composition of any one or clauses 2-5, wherein the    therapeutic agent binds non-specifically to the sulfated or    sulfamated polymer.-   9. The composition of clause 1, wherein the gelling agent is one or    more of a cationic amino acid; an anionic amino acid; a hydrophilic    amino acid; and a cationic, anionic, or hydrophilic polypeptide, and    optionally the gelling agent is a cationic oligopeptide comprising    from 2 to 10 amino acids or from 2 to 6 amino acids.-   10. The composition of clause 9, wherein the gelling agent is a    cationic oligopeptide of from 2 to 10 amino acids, optionally from 2    to 6 amino acids.-   11. The composition of clause 9, wherein the gelling agent is a    homopolymer.-   12. The composition of clause 9, wherein the cationic, anionic, or    hydrophilic amino acids are lysine, arginine, glutamic acid,    aspartic acid, glutamine, histidine, asparagine, serine, tyrosine,    or threonine.-   13. The composition of any one of clauses 1-11, wherein the silicate    platelets comprise hydrous sodium lithium magnesium silicate    platelets, such as LAPONITE® platelets.-   14. The composition of any one or clauses 1-13, wherein the    therapeutic agent is an oligopeptide, a polypeptide or a protein.-   15. The composition of any one or clauses 1-14, wherein the    therapeutic agent is a growth factor.-   16. The composition of any one or clauses 1-15, wherein the    therapeutic agent is Fibroblast Growth Factor 2 (FGF-2).-   17. The composition of any one of clauses 1-16, wherein:    -   a. the ratio of therapeutic agent to the sulfated or sulfamated        polymer and/or to the gelation agent ranges from 1:16000 to 1:1        by weight, from 1:8000 to 1:3 by weight, or from 1:1520 up to        1:3 by weight;    -   b. the ratio of the sulfated or sulfamated polymer and/or to the        gelling agent ranges to the silicate platelets ranges from 1:10        to 1:1 by weight, or from 1:10 to 1:2 by weight; and/or    -   c. the concentration of the silicate platelets ranges from 10        mg/mL to 80 mg/mL (1% to 8%), or from 15 mg/mL to 50 mg/mL (1.5%        to 5.0%).-   18. A method of making a composition for delivery of a therapeutic    agent, comprising:    -   a. mixing a gelling agent with a therapeutic agent that is a        binding partner of gelling agent to produce a complex of the        gelling agent and the therapeutic agent; and    -   b. mixing the complex of the gelling agent and the therapeutic        agent with silicate platelets to produce a shear-thinning        hydrogel.-   19. The method of clause 18, wherein the gelling agent is a sulfated    or sulfamated polymer.-   20. The method of clause 19, wherein the sulfated or sulfamated    polymer is a sulfated glycosaminoglycan.-   21. The method of clause 19 or 20, wherein the therapeutic agent    binds specifically to the sulfated or sulfamated polymer.-   22. The method of clause 19 or 20, wherein the therapeutic agent    binds non-specifically to the sulfated or sulfamated polymer.-   23. The method of clause 20, wherein the sulfated glycosaminoglycan    is heparin or heparan sulfate.-   24. The method of clause 23, wherein the therapeutic agent is a    member of the heparin interactome.-   25. The method of clause 18, wherein the gelling agent is one or    more of a cationic amino acid; an anionic amino acid; a hydrophilic    amino acid; and a cationic, anionic, or hydrophilic polypeptide, and    optionally the gelling agent is a cationic oligopeptide comprising    from 2 to 10 amino acids or from 2 to 6 amino acids.-   26. The method of clause 25, wherein the gelling agent is a cationic    oligopeptide of from 2 to 10 amino acids, optionally from 2 to 6    amino acids.-   27. The method of clause 25, wherein the gelling agent is a    homopolymer.-   28. The method of clause 25, wherein the cationic, anionic, or    hydrophilic amino acids are lysine, arginine, glutamic acid,    aspartic acid, glutamine, histidine, asparagine, serine, tyrosine,    or threonine.-   29. The method of any one of clauses 25-28, wherein the gelling    agent is a poly(glutamic acid) or poly(aspartic acid) oligomer of    from 2 to 10, or from 2 to 6 amino acids.-   30. The method of any one of clause 18-29, wherein the silicate    platelets comprise hydrous sodium lithium magnesium silicate    platelets, such as LAPONITE® platelets.-   31. The method of any one or clauses 18-30, wherein the therapeutic    agent is an oligopeptide, a polypeptide or a protein.-   32. The method of any one or clauses 18-30, wherein the therapeutic    agent is a growth factor.-   33. The method of any one or clauses 18-30, wherein the therapeutic    agent is Fibroblast Growth Factor 2 (FGF-2).-   34. The method of any one or clauses 18-33, wherein:    -   a. the ratio of therapeutic agent to the gelling agent, or to        the sulfated or sulfamated polymer ranges from 1:16000 to 1:1 by        weight, from 1:8000 to 1:3 by weight, or from 1:1520 up to 1:3        by weight;    -   b. the ratio of the gelling agent, or the sulfated or sulfamated        polymer to the silicate platelets ranges from 1:10 to 1:1 by        weight, or from 1:10 to 1:2 by weight; or    -   c. the concentration of the silicate platelets ranges from 10        mg/mL to 80 mg/mL (1% to 8%), or from 15 mg/mL to 35 mg/mL (1.5%        to 5.0%).-   35. The method of any one or clauses 18-33, wherein:    -   a. the ratio of therapeutic agent to the gelling agent, or to        the sulfated or sulfamated polymer ranges from 1:16000 to 1:1 by        weight, from 1:8000 to 1:3 by weight, or from 1:1520 up to 1:3        by weight;    -   b. the ratio of the gelling agent, or the sulfated or sulfamated        polymer to the silicate platelets ranges from 1:10 to 1:1 by        weight, or from 1:10 to 1:2 by weight; and    -   c. the concentration of the silicate platelets ranges from 10        mg/mL to 80 mg/mL (1% to 8%), or from 15 mg/mL to 50 mg/mL (1.5%        to 5.0%).-   36. A method of administering a therapeutic agent to a patient,    comprising administering a shear-thinning therapeutic hydrogel    according to any one of clauses 1-17 to a patient.-   37. The method of clause 36, wherein the shear-thinning therapeutic    hydrogel is administered parenterally at a location in the patient    of an injury or defect, to promote tissue growth, differentiation,    and/or repair of the injury or defect.-   38. A method of inducing angiogenesis in a patient, comprising    administering to a patient an amount of the composition of clause    1-17 effective to induce angiogenesis in a patient, wherein the    therapeutic agent is an angiogenic agent.-   39. The method of clause 38, wherein the angiogenic agent is chosen    from: erythropoietin (EPO), basic fibroblast growth factor (bFGF),    acidic fibroblast growth factor (aFGF), fibroblast growth factor-2    (FGF-2), granulocyte colony stimulating factor (G-CSF), granulocyte    macrophage colony stimulating factor (GM-CSF), hepatocyte growth    factor (HGF), insulin-like growth factors 1 and 2 (IGF-1 and IGF-2),    placental growth factor (PIGF), platelet derived growth factor    (PDGF), stromal derived factor 1 alpha (SDF-1 alpha), and vascular    endothelial growth factor (VEGF), angiopoietins (Ang 1 and Ang 2),    matrix metalloproteinase (MMP), delta-like ligand 4 (D114), and    class 3 semaphorins (SEMA3s), among the others.-   40. A method of filling a soft tissue in a patient, comprising    injecting or implanting in a patient the composition of any one of    clauses 1-17.

While the present invention is described with reference to severaldistinct embodiments, those skilled in the art may make modificationsand alterations without departing from the scope and spirit.Accordingly, the above detailed description is intended to beillustrative rather than restrictive.

We claim:
 1. A shear-thinning therapeutic composition, comprising:silicate platelets; a gelling agent non-covalently linking the plateletsto form a shear-thinning composition; and a therapeutic agent that bindsnon-covalently to the gelling agent and that is complexed non-covalentlywith the gelling agent, wherein the gelling agent is a sulfated orsulfamated polymer, a sulfated or sulfamated polysaccharide, a sulfatedor sulfamated glycosaminoglycan, an amino acid, a cationic polypeptide,an anionic polypeptide, or a hydrophilic polypeptide.
 2. The compositionof claim 1, wherein the gelling agent is heparin or heparan sulfate. 3.The composition of claim 2, wherein the therapeutic agent is a member ofthe heparin interactome.
 4. The composition of claim 1, wherein: theratio of therapeutic agent to the gelling agent ranges from 1:16000 to1:1 by weight, from 1:8000 to 1:3 by weight, or from 1:1520 up to 1:3 byweight; the ratio of the gelling agent to the silicate platelets rangesfrom 1:10 to 1:1 by weight, or from 1:10 to 1:2 by weight; and/or theconcentration of the silicate platelets ranges from 10 mg/mL to 80 mg/mL(1% to 8%), or from 15 mg/mL to 50 mg/mL (1.5% to 5.0%).
 5. Thecomposition of claim 1, wherein the gelling agent is one or more of acationic amino acid; an anionic amino acid; a hydrophilic amino acid;and a cationic, anionic, or hydrophilic polypeptide.
 6. The compositionof claim 5, wherein the gelling agent is a cationic oligopeptidecomprising from 2 to 10 amino acids or from 2 to 6 amino acids.
 7. Thecomposition of claim 1, wherein the silicate platelets comprise hydroussodium lithium magnesium silicate platelets.
 8. The composition of claim1, wherein the therapeutic agent is an oligopeptide, a polypeptide or aprotein.
 9. The composition of claim 1, wherein the therapeutic agent isa growth factor, such as Fibroblast Growth Factor 2 (FGF-2).
 10. Amethod of making a composition for delivery of a therapeutic agent,comprising: mixing a gelling agent with a therapeutic agent that is abinding partner of the gelling agent to produce a complex of the gellingagent and the therapeutic agent; and mixing the complex of the gellingagent and the therapeutic agent with silicate platelets to produce ashear-thinning hydrogel, wherein the gelling agent is a sulfated orsulfamated polymer, a sulfated or sulfamated polysaccharide, a sulfatedor sulfamated glycosaminoglycan, an amino acid, a cationic polypeptide,an anionic polypeptide, or a hydrophilic polypeptide.
 11. The method ofclaim 10, wherein the gelling agent is a sulfated or sulfamated polymer,a sulfated or sulfamated polysaccharide, or a sulfated or sulfamatedglycosaminoglycan.
 12. The method of claim 11, wherein the gelling agentis heparin or heparan sulfate and/or the therapeutic agent is a memberof the heparin interactome.
 13. The method of claim 10, wherein thegelling agent is one or more of a cationic amino acid; an anionic aminoacid; a hydrophilic amino acid; and a cationic, anionic, or hydrophilicpolypeptide.
 14. The method of claim 13, wherein the gelling agent is acationic oligopeptide comprising from 2 to 10 amino acids or from 2 to 6amino acids.
 15. The method of claim 10, wherein the silicate plateletscomprise hydrous sodium lithium magnesium silicate platelets.
 16. Themethod of claim 10, wherein the therapeutic agent is an oligopeptide, apolypeptide or a protein, such as a growth factor, such as FibroblastGrowth Factor 2 (FGF-2).
 17. The method of claim 10, wherein: the ratioof therapeutic agent to the gelling agent ranges from 1:16000 to 1:1 byweight, from 1:8000 to 1:3 by weight, or from 1:1520 up to 1:3 byweight; the ratio of the gelling agent to the silicate platelets rangesfrom 1:10 to 1:1 by weight, or from 1:10 to 1:2 by weight; or theconcentration of the silicate platelets ranges from 10 mg/mL to 80 mg/mL(1% to 8%), or from 15 mg/mL to 35 mg/mL (1.5% to 5.0%).
 18. The methodof claim 10, wherein: the ratio of therapeutic agent to the gellingagent, or to the sulfated or sulfamated polymer ranges from 1:16000 to1:1 by weight, from 1:8000 to 1:3 by weight, or from 1:1520 up to 1:3 byweight; the ratio of the gelling agent, or the sulfated or sulfamatedpolymer to the silicate platelets ranges from 1:10 to 1:1 by weight, orfrom 1:10 to 1:2 by weight; and the concentration of the silicateplatelets ranges from 10 mg/mL to 80 mg/mL (1% to 8%), or from 15 mg/mLto 50 mg/mL (1.5% to 5.0%).
 19. The method of claim 10, wherein theshear-thinning therapeutic hydrogel is administered parenterally at alocation in the patient of an injury or defect, to promote tissuegrowth, differentiation, and/or repair of the injury or defect.
 20. Amethod of inducing angiogenesis in a patient, comprising administeringto a patient an amount of the composition of claim 1 effective to induceangiogenesis in a patient, wherein the therapeutic agent is anangiogenic agent.
 21. The method of claim 20, wherein the angiogenicagent is one or more of: erythropoietin (EPO), basic fibroblast growthfactor (bFGF), acidic fibroblast growth factor (aFGF), fibroblast growthfactor-2 (FGF-2), granulocyte colony stimulating factor (G-CSF),granulocyte macrophage colony stimulating factor (GM-CSF), hepatocytegrowth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-likegrowth factor 2 (IGF-2), placental growth factor (PIGF), plateletderived growth factor (PDGF), stromal derived factor 1 alpha (SDF-1alpha), vascular endothelial growth factor (VEGF), angiopoietin 1 (Ang1), angiopoietin 2 (Ang 2), matrix metalloproteinase (MMP), delta-likeligand 4 (DI14), or a class 3 semaphorin (SEMA3).
 22. A method offilling a soft tissue in a patient, comprising injecting or implantingin a patient the composition of claim 1.